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1 | Download: File > Download > Microsoft Excel or Have a copy in your Drive: File > Make a copy > Choose Location | |||
2 | Khan Academy MCAT Videos (Drive folder in case KA takes them down from Youtube) | |||
3 | Unit | Chapter | Video Link | Transcript |
4 | Critical Analysis And Reasoning Skills Practice Questions | Critical Analysis And Reasoning Skills Tutorial | CARS overview | Hello, my name is Jennifer Riley, and I'm a cognitive scientist who studies text comprehension and reasoning. The goal of this video is to help you better understand the new section of the MCAT, called the critical analysis and reasoning skills section. Its main purpose is to figure out how well you can understand and reason about the things you read. In this part of the test, you'll read nine passages. Each one is about 500 to 600 words long, and you'll usually be asked between five to seven questions about each passage. In total, you'll go through 53 questions in 90 minutes. That means you have about 10 minutes per passage, on average. In terms of the topics that you'll be reading about, approximately half of these passages will come from the humanities, such as literature, philosophy or ethics. The other half will come from social sciences, such as psychology, sociology or economics. For example, one of the passages that's in the Khan Academy MCAT collection discusses how people negotiate deals with one another, and when they believe they are getting a fair deal. Another passage is about how computers, rather than just being tools used by humans, might actually be changing the way that humans think. The passages come from a lot of different areas, and are meant to stretch your mind a bit. You'll probably find that the topics of these passages will be unfamiliar. You're not expected to already know about any of these topics. Sometimes the writing styles will be complicated. Some words may be new to you, and some passages may be tough to understand. Just remember, everything that you need to know to answer the questions will be in the passages. You're not expected to know any background information. In fact, to do well, you really need to just focus on the passage, and not information that you already know from elsewhere. There are three types of questions you'll be asked to answer in this section. The first type is called foundations of comprehension. These questions mainly ask you to answer questions about the author's intended message. They'll ask you about the overall idea, or about why the author used specific words or phrases, or why the author organized the passage in a specific way. The second category is called reasoning within the text, and these questions mainly ask you to think about the reasoning within an author's argument, such as, what claim is an author trying to support with a piece of evidence? Or, is an argument flawed? The third category is called reasoning beyond the text. Questions in this category ask you to apply ideas from the passage to new situations, or to think about how the author's main message would change if there were new information to consider. This new information will be given to you in the question. About a third of the questions will come from each category. You should expect about 30% foundations of comprehension questions, 30% reasoning within the text questions, and 40% reasoning beyond the text questions. People often wonder how the skills that are tested by the critical analysis and reasoning skills section will ever be useful for a future physician. These questions are on the MCAT because doctors need to be able to reason through a lot of clinical information. For example, these skills are needed when you need to analyze information to come up with a treatment plan that makes sense for a patient, and also so that you can explain your reasoning to others, including the patient, family members, or other members of your medical team. For more information on this section of the test, and on each of the question types, be sure to see the other videos in this section, which I hope will help you, too. |
5 | Critical Analysis And Reasoning Skills Practice Questions | Critical Analysis And Reasoning Skills Tutorial | Foundations of comprehension | Hi, in this video I'm going to tell you about foundations of comprehension questions. These are one of three types of questions on the critical analysis and reasoning skills section of the MCAT. These questions are designed to test your basic understanding of the passage. These questions can take many forms, but many of these will be familiar to you from other tests. Some common questions will ask you about the main idea or theme of the passage, about the intended meaning of specific words or phrases, or why the author organized or wrote a passage in a specific way. Let's start with a quick example of a main idea question. Which of the following best captures the main goal of the passage? First of all, where it says, "the main goal," we could replace that phrase with the "key idea," "core theme," "central purpose." All of these are asking about the main idea of the passage. I could also replace "passage" with "final paragraph," or "third paragraph," and it's still the same type of question. All I want you to notice here is that the question wants you to be able to recognize a good summary or paraphrase in the main idea that the author is saying. Another similar type of question could ask you to identify an author's main claim or position. The author will often state this for you in a passage using a thesis statement that might start with words like, "I believe," or "It is clear that," or "My main point is." This usually comes in an early section. For example, take a look at this statement: "Every student deserves one year off to explore the world." This statement is important because it tells you what the author believes. It serves as the author's thesis statement and foreshadows what they are going to argue. By saying "deserves," the author is suggesting that every student who wants to take a year off to explore the world should be able to do so. This can also be seen as a claim that the author is making. A claim is a statement that gives you the author's position, point of view, or perspective on a topic. You could also be asked what conclusion the author is trying to draw. This is yet another way of asking you about the main point that the author is trying to make. When answering these questions, one great place to look is at the end of the passage to see if the author attempts to offer a clear conclusion as part of the closing section. Examples of these kinds of questions include, "Which of the following phrases most accurately "captures the author's theme?" "Based on the passage, the author most likely "believes that." "Which of the following sentences best represents "the author's conclusion?" Some questions will ask you to infer the author's attitude or perspective on a topic by considering their particular words or phrases. For example, if an author writes, "How many people died in the Vietnam War? "A novice might count up the bodies on the battlefield. "However, expert analysis shows us that deaths off "the battlefield far outnumber deaths on the battlefield." A question about this example might ask, "Which approach to assessing the death toll "in Vietnam does the author favor?" The author uses the word "novice," and "expert," to suggest that the expert way is better. Sometimes the words themselves come loaded with meaning. We call that connotative language. The author can reveal their perspective or attitude toward information by using loaded adjectives or adverbs, like "evil," "valuable," "unfortunately," or "rightly." Some questions may require that you use the absence or presence or such words to infer whether the author seems to be neutrally and objectively conveying factual information versus stating their opinion, or revealing a bias about the issue. Some questions will ask you to determine the intended meaning of specific words or phrases. Sometimes the author will use unfamiliar words or terms, and you'll need to figure out their meaning from the rest of the passage. Other times, the author will use familiar terms, and you'll need to decide the precise way they are being used. An example of this question would be, "What does the author "mean by the phrase "medical interventions"?" If in the passage, the author introduces the idea of medical interventions, and then gives you examples of helmets, seat belts, vaccines, talk therapy, massages, home remedies, yoga, medications, and surgeries, then you get the sense that the author's definition of medical intervention is really pretty broad. Instead, if the author talks about medical interventions and then just discusses different types of antibiotics, then that would be a more narrow definition of the term. For other foundation questions, you'll need to pay attention to the author's use of signal words, and to consider the passage structure. One example that you might use signal words or text structure to answer is, "What is the primary purpose of paragraph 3?" The author can include many kinds of signal words that you could use to help answer this question. These can include words and phrases like "importantly," "the only thing that matters is," or "for example." This can help you identify basic information like thesis statements, main ideas, and examples. Phrases like "by the way," or "in a few cases," can help signal a minor point, departure, or digression from the main theme. Phrases like "in addition to," "therefore," and "consequently," can help you follow an author's argument. Considering the structure of the passage can also help you in similar ways. For example, two common text structures, point counterpoint, and compare and contrast, can also help you to identify distinctions being made by the author. A point counterpoint text is almost like a discussion between two people. The author goes back and forth laying out points from two different sides. A compare and contrast text will generally first discuss the commonalities between two things, and then highlight the differences. Noticing when passages have these structures will help you know where to look to see how positions or concepts might differ. Phrases like, "on the other hand," "in contrast to," "but," or "however," are likely to appear in these kinds of texts, and they can help you to identify distinctions that the author's trying to make. An example of a question that you might use these ideas to answer is, "According to the author, what are "two perspectives on this issue?" Other common structures include listing of ideas, or a chronological structure, where the author walks you through events in the order they happened. In these cases, it's often helpful to look for words like, first, second, or third, that might help you to identify how many different points are being made; or words like "next," and "and then," that might help you follow the chain of events. Another common text structure is the cause effect structure, where the author first lays out a set of conditions or factors, and then discusses how effects or consequences result from them. In these passages, you should look for causal words like "because," or "due to." An example of a question that you might use these ideas to answer is, "What is the primary cause of the problem "as implied by the passage?" If there's a specific phrase that you are asked about, then it can be helpful to consider how the passage is organized, where that phrase appears, and that can give you a clue of what its purpose might be. Another final type of example is that you may be asked to interpret rhetorical devices. These are devices that the author uses as a way to convey their message. Rhetorical devices include the signal words discussed above like "however," and "on the other hand," but they also use things like repetition or parallelism. Repetition and parallelism are when words or sentence structures are repeated. These are examples of literary devices that may be used by the author to draw attention to particular phrases. The author may also use other literary devices such as metaphor, sarcasm, allegory, or symbolism. Metaphor is when the author compares one thing to another. "Jobs are prisons." Sarcasm is when the opposite of what is stated is meant. "Dental work is a joy." Allegory is when the author relates a narrative or story that's really meant to be understood as being about something else, with the characters being meant to personify abstract ideas. In all of these cases, the author is not expecting their words to be taken literally, and questions may ask you how the author is intending their words to be interpreted. An example of a question asking you about the author's use of a literary device is, "The author makes use of sarcasm in this passage for what purpose?" A closely related idea is that of symbolism. Symbolism is when the author uses an object or concept to represent something else. An author may describe an episode in which a character sees a beautiful flower that's fighting to grow through the cracks of a grimy city sidewalk. In this case, the author may mean that the character actually sees the flower, but may also be using the image to convey a message of beauty in unexpected places, which may be a theme for the work. An example of a question about the author's use of this device would be, "Which of the following is implied "by the use of imagery about the flower?" Similar to word choice, literary devices and figures of speech like metaphors can also reveal an author's position or attitude on a topic. They can color the author's message by using more or less pleasing examples. For example, comparing a political candidate to Robin Hood is likely to suggest a more positive view of the candidate than comparing the candidate to Hitler. An example of a question that asks you about this idea is, "It can be inferred from the author's tone that they believe "which of the following?" I hope this video has given you a good idea of what many questions in the foundations of comprehension category will ask you about. To better understand these types of questions, be sure to try some practice items, and check out the other videos in this section. |
6 | Critical Analysis And Reasoning Skills Practice Questions | Critical Analysis And Reasoning Skills Tutorial | Reasoning within the text | The purpose of this video is to help you understand the reasoning with the text questions. These are one of three categories of questions on the critical analysis and reasoning skills section of the MCAT. The key feature of these questions is that they ask you to examine the arguments being made by the author. These questions will direct your attention to arguments, claims, conclusions, or pieces of evidence that are presented in the passage. They will ask you analyze and evaluate the author's argument in some way. There are two major types of these questions. The first type includes structural questions that require you to identify how the author is trying to relate various ideas in the passage. These questions ask you to recognize which claim a particular example or piece of evidence is intended to support. The second type include evaluative questions that require you to be critical, and consider flaws or weaknesses in the author's argument or evidence. Anytime you're being asked how the author provides support or evidence for their position or claim, it's a good clue that it's a reasoning within the text question. Here's an example of the first type of question that asks you to identify the relation between claims and evidence in a passage. "Which of the following quotes is presented as evidence for the author's position?" To answer this question, there are two main things you need to do. The first is that you need to identify the author's position. Then, the second thing you need to do is find each of the quotes in the response options within the passage, and determine which of them is being used to support the author's position. Finding the correct answer to this question requires identifying how the author uses each piece of information as part of an argument. In this example question, the response options could include four quotes that actually appear in the text, and you will need to decide between them. However, it's important to note that this is not true of all reasoning within the text questions. Sometimes you'll need to eliminate answer options that don't accurately represent statements or ideas from the passage. Here's another example of this type of question. "Which of the following passage assertions is presented as evidence that computers are affecting people's conception of the mind?" To answer this question, you would first need to eliminate any answer options that do not accurately summarize statements actually made in the passage. Then, for ideas that do appear in the passage, you'd need to determine how the author presents each of them in relation to the specific claim that computers are affecting people's conception of the mind. Here's a third example of this kind of question. "Which conclusion does the author use this example to support?" Instead of presenting you with a specific claim as part of the question, and asking you to identify the evidence that the author provides for it, this question does the reverse. It presents you with a piece of evidence, and asks you to choose which claim the author uses it to support. The second major type of reading within the text question requires you to evaluate, be critical, and consider flaws or weaknesses with the author's argument. For example, as you are reading, you may notice that an author includes statements that seem to be inconsistent with each other. Other times you may notice that an author is making conclusions that seem unjustified. Sometimes connections that seem fine when you first read them, won't seem as strong when you examine them closely. Suppose you read a passage about Nepal that includes these sentences. "Nepal is an underdeveloped country that is one of the most disasterprone in the world. In Nepal, poverty drives people to live in highrisk areas which makes them vulnerable to disasters. Disasters in Nepal affect a large number of people by destroying their houses, productive lands, other personal assets, and livelihoods. Hence poverty is both a cause and a consequence of disasters in underdeveloped countries." A possible question asking you to evaluate the author's reasoning would be, "What is a weakness in the argument the author makes to support their conclusion about the relation of poverty to disasters?" Option A, "The author fails to explain how people are affected by disasters." Option B, "The author assumes that the situation in Nepal will generalize to all underdeveloped countries." Option C, "The author fails to consider the role of poverty in causing disasters." And option D, "The author fails to consider the role that disasters play in causing poverty." If you read back through the excerpt, you can see that the author does consider issues A, C, and D. Answering this question requires noticing that although all prior sentences are concerned only with Nepal, in the final conclusion, the author makes a general statement about the causal relation between poverty and disasters for underdeveloped countries. The author is assuming whatever is true of Nepal would generalize to other underdeveloped countries. Thus, option B correctly identifies a weakness in the reasoning within the text. Sometimes a question will ask you to identify an unstated assumption that the author is making, such as, "What assumption does the author make about gun violence?" For example, if a passage claimed, "Raising the price of bullets will lower gun violence," a key assumption implied by that statement is that people who commit gun violence aren't willing to buy bullets at a higher price. Whether the assumption seems like a reasonable one or not is irrelevant to answering this kind of question. Assumptions can be facts that few would question, or can be highly controversial and unsupported ideas. What's important for answering this kind of question is that there's something the author did not explicitly say, but that needs to be true in order for the author's conclusion to make sense. Sometimes an author will provide irrelevant, subjective, or biased information to try to support their ideas. It's important to consider whether the evidence is actually relevant for the point that the author is trying to make. An example of a question about this is, "Which of these examples is irrelevant for the claim that sugar is unhealthy?" It's also important to consider the kinds of information and sources that the author cites to support their point of view. Does the evidence seem to be subjective? Is it based in fact? Is it possible to objectively verify? An example of a question about this, is "Which of the following statements is an opinion and not a fact?" Finally, it's especially important to remember that the reasoning within the text questions want you to evaluate the strength of an author's argument or reasoning in terms of the information presented in the passage. You'll need to be careful not to introduce your own personal opinion on the topic. You might not agree with the position that the author takes, or you might know of some critical information that contradicts statements in the passage, but neither of these are important. The key is to just base your responses on the information as provided in the passage, and analyze it on that basis alone. To better understand these types of questions, be sure to try some practice items, and check out the other videos in this section. |
7 | Critical Analysis And Reasoning Skills Practice Questions | Critical Analysis And Reasoning Skills Tutorial | Reasoning beyond the text | The purpose of this video is to help you understand the Reasoning Beyond the Text questions a bit better. These are one of the three main categories of questions on the Critical Analysis and Reasoning Skills section of the MCAT. These questions should be the easiest to spot, because you'll be given something new to think about as part of the question, like a new situation, or a new piece of information, or new examples. There are two main types of Reasoning Beyond the Text questions. One type asks you to apply the concepts in the passage to brand new situations. The other asks you to integrate a new piece of information with the information that was given to you in the passage, to see how that new information would effect the overall interpretation. Let's start with the first type of question, which asks you to apply the passage concepts to new situations. Here is an example. Someone who agreed with President Wilson's explanation of the need for a general association of nations, would be most likely to also approve of which of the following hypothetical options? The question stem starts by referring to someone, as a way of introducing a new person who is not mentioned in the passage. When the question introduces a new person or context, this is a clue that this will be a Reasoning Beyond the Text question. The question also says you're being asked to think about hypothetical options. Hypothetical means that something exists as a possibility, but that it may or may not be true in reality. This phrase is another clue that you're being asked to go beyond what the text says. You might also be asked to consider new possibilities, alternatives, options, or proposals. To answer this particular question, you need to focus your attention on what you believe the key parts of President Wilson's explanation are. And then, look for an answer that preserves the role for those key parts. When you get a question like this, go through each of the options, and figure out which new example or options best fits the ideas stated in the passage. Other questions of this type may just give you a new set of scenarios to consider, and ask you which is most consistent with the point made by the author. Here are some examples of questions of this type. Which new example is most consistent with the author's definition of art? Which new situation best captures the relationships between cats and their owners as described in the text? Which of these proposed policies would you expect to be the most successful, based in the author's argument? Another way that a question can ask you to go beyond the text, is by giving you new information to consider. This could be a fact that was not mentioned in the passage, or new information that came to light after the passage was written. Let's take a look at an example of this type of question, which asks you to integrate new information. If it were known that Neanderthals and Homo Sapeins coexisted, but they lived in geographic isolation from one another, how would this affect the conclusions reached by the author? This question starts with the word, if, which is a signal that you'll be asked to consider a new condition. Other questions might start with similar words, like suppose, or assume, or imagine, or they might start with, what if. In each case, these words are generally used to give you new information that was not mentioned in the passage. Once you read the new information, you'll need to assess how that information might affect the arguments made in the passage. May of these questions are asking you to think about whether the new information is consistent, or inconsistent with the reasoning in the passage. Does the new information provide additional support for the author's argument? Or, does it conflict with evidence that is cited in the text? Or does it contradict a conclusion that the author reached? Does it require you to refine or specify part of the argument made by the author? For these questions, it's especially important to remember that the right answer will be one that can be justified by considering something in the passage. Always remember to answer using only the information provided in the passage in question, and not based on outside information that you may have about the topic. You should also be sure to avoid using your own personal opinion. Here are some other examples of these types of questions. Imagine that humans had no thumbs. How would this affect the author's argument? Which of the following newly discovered pieces of evidence would go against the theory developed in the passage? Suppose a new species was found that could live underwater without light. What impact would that have on the definition of life proposed by the author? The basic concept that the MCAT wants students to understand, is that the inferences and conclusions that are supported by the passages are all subject to change, and need to be adjusted as new information bubbles up. This is an important skill, because doctors need to continually update their understanding of diseases and treatments as they get new information. To better understand these types of questions, be sure to try some practice items, and check out the other videos in this section. |
8 | Critical Analysis And Reasoning Skills Practice Questions | Critical Analysis And Reasoning Skills Tutorial | Worked example: Living in a rational society | The purpose of this video is to show you how I approach reading and answering questions on an example Critical Analysis and Reasoning Skills passage. The name of this passage is "Living in a rational society." I'm gonna read through the passage first, and when I notice important sentences or signal words, I'll let you know that I'm highlighting them. "The rationalizing of society can be conceptualized as the pursuit of efficiency, predictability, calculability, and control through technology. But rational systems inevitably spawn a series of irrationalities that result in the compromising and perhaps even the undermining of their rationality." Okay, so that first sentence seems pretty important because it gives a definition for the term "Rationalizing of Society" which is also mentioned in the title of this passage. So I'm gonna highlight that sentence. I'm gonna wait and see if the point about irrationalities made in the second sentence seems important after I read some more. "Fast food restaurants, which epitomize the rational model, prefer the fastest means of getting from a hungry state to a sated one, without surprises, at low cost and in a carnival like setting, suggesting that fun awaits the consumer at each visit. The wholesomeness of the food seems an insignificant consideration. Whereas in the past working people were prepared to spend up to an hour preparing dinner, they are now impatient if a meal is not on the table within ten minutes. For their part, some fast food restaurants have developed chairs that become uncomfortable after about twenty minutes to ensure that diners do not stay long." So here the author signals that fast food restaurants are the epitome, or ideal example of a rational system. I'm gonna highlight that part of the first sentence. Then the rest of this section seems to be explaining which features of fast food restaurants make them such a good example. "Fast food restaurants have preferentially recruited adolescent help; at least until recently, because this age group adjusts more easily than adults do to surrendering their autonomy to machines, rules and procedures. Few skills are required on the job, so workers are asked to use only a minute portion of their abilities. This policy is irrational from the standpoint of the organization, since it could obtain much more from it's employees for the money, however negligible it pays them. These minimal skill demands are also irrational from the perspective of the employees, who are not allowed to think or to respond creatively to the demands of the work. These restrictions lead to high levels of resentment, job dissatisfaction, alienation, absenteeism and turnover among workers in fast food franchises. In fact, these businesses have the highest turnover rate of any industry in the U.S. The entire workforce of the fast food industry turns over three times in a year. Although the simple repetitive nature of the work makes it easy to replace those who leave, the organization would clearly benefit from keeping employees longer. The cost of hiring and training are magnified when the turnover rate is extraordinarily high." So these two paragraphs tell us more about the features of fast food restaurants that are consistent with the rational model. But here we also see the theme of irrationality emerge. So I'm gonna go back and highlight that theme that we saw mentioned in the second sentence. I'm also going to highlight where evidence for that theme is mentioned here. Finally, we are told what some negative consequences of the rational model seem to be. The first sentence of the fourth paragraph summarizes these negative effects. So I'm gonna highlight that first sentence. Now let's continue with the passage. "The application of the rational model to the housebuilding process in the 1950's and 60's led to suburban communities consisting of nearly identical structures. Indeed, it was possible to wander into the residence of someone else and not to realize immediately that one was not at home. The more expensive developments were superficially more diversified, but their interior layouts assumed residents who were indistinguishable in their requirements. Furthermore the planned communities themselves looked very similar. Established trees are bulldozed to facilitate construction, in their place a number of saplings, held up by posts and wire are planted. Streets are laid out in symmetrical grid patterns with such uniformity suburbanites may well enter the wrong subdivision or become lost in their own." So in this section the author turns to a new example: House building. I'm gonna highlight the first sentence to remind me where this new topic starts. Then the remainder of this section seems to be describing how elements of the rational model can be seen in this new context. Let's see where the passage goes next. "Many of Steven Spielberg's films are set in such suburbs. Spielberg's strategy is to lure the viewer into this highly repetitive world and then to have a completely unexpected event occur. For example, the film Poltergeist takes place in a conventional suburban household in which evil spirits ultimately disrupt the sameness. The spirits first manifest themselves through another key element of the homogeneous society, the television set. The great success of Spielberg's films may be traceable to a longing for some unpredictability, even if it is bizarre and menacing, in increasingly routinized lives." At first it's unclear what Steven Spielberg's films would have to do with the rest of the passage. It seems like a digression or a departure from the author's main point. But then the last sentence is important because it gives us a reason why the author goes into this example. I'm gonna highlight that sentence. It's used to show how routinized lives, as dictated by the rationalized model, may not be satisfying for people. Finally we see the reference for this passage, "Adapted from G. Ritzer, The McDonaldization of Society Copyright 1993 by Pine Forge Press. Question one. The author's argument suggests that the primary motive of employers who make humans work with machines is to: A. Improve the quality of their products. B. Reduce the cost of wages and benefits. C. Avoid seeming to be behind the times. D. Increase the uniformity of procedures. This question is asking you to summarize or paraphrase what the author suggests is the primary motive of employers who make humans work with machines. Because it's asking you to summarize a specific phrase or idea from the text, this is a "Foundations of Comprehension" question. Since the question is about employers, this suggests that the answer to the question's going to be found in the first part of the text, about the fast food industry rather than the second part of the text about housebuilding. Option A suggests that employers are motivated to improve the quality of their products. In paragraph Two, the only part of the text that comes close to discussing quality is the sentence that says "The wholesomeness of the food seems an insignificant consideration.." This suggests that the author believes quality of a product is not an important motivation. Thus it doesn't appear that option A is a good answer. Option B suggests that employers are motivated to reduce the costs of wages and benefits. Although the author mentions that fast food restaurants offer products at low cost, there's no discussion about trying to reduce the cost of wages and benefits with machines. So it doesn't appear that option B is a good response. Option C says that employers are motivated to keep up with the times, but there's nothing in the passage that suggests that employers are trying to stay current, or seem innovative or cutting edge. So it doesn't seem that option C is a good response. Option D suggests that the employer is motivated to increase uniformity. The opening sentence mentions control through technology. In the second paragraph the author cites the desire to avoid surprises for the consumer. In the third paragraph the author cites employer preference to hire employees that are comfortable with surrendering their autonomy to machines, rules and procedures. All these examples are consistent with the claim that the employer has control and uniformity of procedures as the main goal in using machines. Thus, there is some evidence in the passage to support option D, and it seems like the best answer. Question two. "A common thread in the discussion of fast food and the discussion of suburban housing is that people today: A. Are increasingly resistant to the regimentation of life. B. Expect their needs to be met at the lowest possible cost. C. Allow themselves to be treated as interchangeable. D. Are unable to discriminate among products that differ in quality." This question is asking you to identify a central theme or idea from the passage. This means it's a "Foundations of Comprehension" question. Option A says that people are becoming increasingly resistant to regimentation. Skimming through both the fast food and the housing sections there's nothing suggesting that individuals are resisting regimentation of life, nor that they are increasingly resistant. So it doesn't appear that this is a good response. Option B says that people expect their needs to be met at the lowest possible cost. Although the discussion of the fast food industry suggests that consumers expect their fast food at low cost, there's no discussion of people expecting low cost in suburban housing. So it doesn't appear that option B is a common thread across the two situations. Option C says that people allow themselves to be treated interchangeably. There does seem to be evidence for this being true of the fast food industry. In paragraph three, it describes fast food employees as being willing to surrender their autonomy to machines, rules and procedures and to work in jobs where they are not allowed to think or respond creatively. In paragraph four it says, "the simple repetitive nature of the work makes it easy replace those who leave." Then in paragraph five the passage details a number of ways that people allow themselves to be interchangeable in terms of suburban housing. People live in nearly identical structures, and builders assume residents are indistinguishable. So option C seems to be a good answer. However, before selecting it we should examine whether option D is better. Option D suggests that people are unable to discriminate quality. In the fast food portion of the passage, the author suggests that wholesomeness of food seems an insignificant consideration. However, just because people do not consider the quality of their fast food it doesn't mean they cannot discriminate it. Further, as we read through the suburban housing portion of the passage, we see there's no discussion about quality in home construction. So it doesn't appear that option D is a common thread, and option C seems the be the best answer. Question three. "Information in the passage suggests that a rationalized travel agency would emphasize: A. Planned tours to popular attractions with accommodations at large hotels. B. Computerized systems to provide lowcost customized itineraries. C. Personnel trained to make reservations but with little experience as travelers. D. Procedures that encourage problem solving initiatives by managers." This question introduces a new idea that was not mentioned in the passage. It asks you to imagine a rationalized travel agency. Because you're being asked about a new context, this is a "Reasoning beyond the text" question, which means it wants you to either apply or extrapolate the ideas in the passage to the new situation. Returning to the definition of a rationalized system that we highlighted in the first sentence, we would expect a rationalized travel agency to emphasize the pursuit of efficiency, predictability, calculability and control through technology. We would expect standardization for a large group of consumers. Option A offers planned tours to popular destinations and accommodation in large hotels. This fits well with the rationalized approach, so option A would seem to be a good answer. Option B suggests the use of computerized systems to produce low cost customized itineraries. The use of computers is obviously a form of technology which is mentioned as an aspect of a rational model. However, customized itineraries would represent the opposite of the rational model, because they would offer individual variations rather than predictability and sameness. So option B does not appear to be a good response. Option C suggests that employees might be trained to make reservations, but have little travel experience. The passage does not discuss using employee background including prior experiences or lack of prior experiences as part of personnel decisions under the rational model. So it doesn't appear that option C is a good response. Option D says that the rationalized travel agency would emphasize problem solving among managers. However, passage says that under the rationalized model employees are not allowed to think or respond creatively to the demands of work. It does not discuss different practices for senior level employees such as managers. Thus there's no support in the passage for option D. Option A is the answer that has the most support from the passage. Question four: "Suppose that the employee responses to working conditions in fast fast food franchises paragraph four also apply to entrylevel assembly line workers. In light of this information, the author's main point in mentioning these responses is: A. Weakened, since the fast food industry is not unique in suppressing creativity. B. Weakened, since the monotony of work is not necessarily related to employee dissatisfaction. C. Strengthened, since predictability and employee turnover are associated in another context. D. Strengthened, since low wages and job dissatisfaction are associated in another context." This question starts with the word "suppose". That is a good clue that this is a "Reasoning beyond the text" question, which means that it wants you to apply or extrapolate the ideas in the passage to a new situation. Or to think about how new information would affect the ideas in the passage. The passage uses the fast food industry as an example. The author described employee responses to working conditions in the fast food industry in the sentence we highlighted at the start of paragraph four. "These restrictions lead to high levels of resentment, job dissatisfaction, alienation, absenteeism and turnover among workers in fast food franchises." The term "restrictions" in this sentence refers to the previous sentence where it says that employees are not allowed to think or respond creatively. If this pattern also seen in another context, such as entrylevel assembly line workers, then the authors main point would be strengthened, not weakened as proposed in options A and B. So those do not seem like good answers. Both options C and D do state that the authors main point would be strengthened. Option C describes the authors main point as being a connection between predictability and turnover. This is consistent with the ideas in the highlighted sentence, suggesting that turnover is related to being restricted to only using a standard set of routines. So there is support for option C. Option D describes the authors main point as being a connection between low wages and dissatisfaction. However, the passage does not discuss low wages as one of the reasons why employees may be dissatisfied. Thus, option D does not seem like a good answer, and option C is a better answer. |
9 | Critical Analysis And Reasoning Skills Practice Questions | Critical Analysis And Reasoning Skills Tutorial | Worked example: The happy American | The purpose of this video, is to show you how I approach reading and answering questions on an example of critical analysis and reasoning skills passage. The name of this example passage, is "The Happy American". I'm going to read through the passage first and after each major section of the passage, i'll think about what words or sentences seem important to me. I'll let you know when I'm highlighting words or sentences. Let's start reading this passage now. Americans are a “positive” people. This is their reputation as well as their selfimage. In the wellworn stereotype, they are upbeat, cheerful, and optimistic. Who would be churlish enough to challenge these happy features of the American personality? Take the business of positive “affect,” which refers to the mood they display to others through their smiles, their greetings, their professions of confidence and optimism. Scientists have found that the mere act of smiling can generate positive feelings within us, at least if the smile is not forced. In addition, recent studies show that happy feelings flit easily through social networks, so that one person’s good fortune can brighten the day even for only distantly connected others. Furthermore, psychologists agree that positive feelings can actually lengthen our lives and improve our health. People who report having positive feelings are more likely to participate in a rich social life, and social connectedness turns out to be an important defense against depression, which is a known risk for many physical illnesses. Okay. So the gist of this first part seems to be that the author is describing Americans as positive people. And the author also gives several reasons why being positive is a good thing. I'm going to just highlight the first two sentences since they seem to capture the main point that Americans seem to be seen as positive. Now let's go back to the passage. It is a sign of progress, then, that economists have begun to show an interest in using happiness rather than just the gross national product as a measure of an economy’s success. Happiness is, of course, a slippery thing to measure or define. Philosophers have debated what it is for centuries and even if they were to define it simply as a greater frequency of positive feelings than negative ones, when they ask people if they are happy, they are asking them to arrive at some sort of average over many moods and moments. So in this passage, the author shifts from talking about positive feelings, to talking about happiness. The author also notes that happiness is hard to measure and define. So, I'm going to highlight that first sentence that shows that the passage moves to talking about happiness. but also that second sentence, saying that happiness is hard to measure and define. Those seem like the author's main points for this second part. Now let's go back to the passage again. Surprisingly, when psychologists measure the relative happiness of nations, they routinely find that Americans are not even in prosperous times and despite their vaunted positivity, very happy at all. A recent metaanalysis of over a hundred studies of selfreported happiness worldwide found Americans ranking only twentythird. Americans account for twothirds of the global market for antidepressants, which happen also to be the most commonly prescribed drugs in the United States. So, this paragraph introduces an interesting wrinkle in the author's argument. Now we hear there is reason to think that American's may not actually be very happy. The author recognizes that this seems in conflict with the general perception of them as positive. And the phrase, "despite their vaunted positivity, they are not very happy at all," shows that the author seems to be aware of this conflict. So I'm going to go back and highlight that first sentence, because it seems pretty important for the author's agument. Now, lets go back to the passage. How can Americans be so surpassingly “positive” in selfimage and stereotype without being the world’s happiest and bestoff people? The answer is that positivity is not so much their condition as it is part of their ideology— the way they explain the world and think they ought to function within it. That ideology is “positive thinking,” by which they usually mean two things. One is the generic content of positive thinking— that is, the positive thought itself— which can be summarized as “Things are pretty good right now, at least if you are willing to see silver linings, and make lemonade out of lemons, etc., and things are going to get a whole lot better.” The second thing they mean by “positive thinking” is this practice of trying to think in a positive way. There is, they are told, a practical reason for undertaking this effort: positive thinking supposedly not only makes us feel optimistic but actually makes happy outcomes more likely. How can the mere process of thinking do this? In the rational explanation that many psychologists would offer today, optimism improves health, personal efficacy, confidence, and resilience, making it easier for us to accomplish our goals. A far less rational theory also runs rampant in American ideology—the idea that our thoughts can, in some mysterious way, directly affect the physical world. Negative thoughts somehow produce negative outcomes, while positive thoughts realize themselves in the form of health, prosperity, and success. For both rational and mystical reasons, then, the effort of positive thinking is said to be well worth our time and attention. So in this final section, the author uses a problem/solution or a question/answer text structure to present us with both a question about the pursued conflict that's forshadowed in the earlier section, and also a possible response. Both the question and the response seem like important points to hightlight. In addition, notice that the author signals to you that there are two parts to the answer. I'm highlighting these signals also. In particular, the phrases, "two things. One" and "The second thing." Finally, in the second part of the answer, the author asks how positive thinking makes happy outcomes more likely, and then offers two explanations. The author's signaled their own opinion about these two explanations by labeling one as "rational" and the other as "far less rational". I'm highlighting these signals, in case i need them later. Finally we see that this excerpt has been adapted from B. Ehrenreich, Brightsided. Copyright 2009 by Metropolitan Books. Now let's look at the first question. According to the passage, positive feelings are: A. Universal B. Hereditary C. Contagious D. Ephemeral The first option suggests positive feelings are universal. So we should look through the passage for some evidence that everyone experiences positive feelings. However, what we find is that there is variability in the amount of positive feelings that people report. So the passage does not seem to support the idea that positive feelings are universal. So we can strike through that answer. The second option, suggests that positive feelings might be hereditary. However the passage does not discuss the idea that positive feelings are either inherited or genetic in their basis. So it doesn't seem that heredity is going to be a good answer either, and we can strike through option B. The third option we need to explore, is whether positive feelings might be contagious. In the passage, we're looking for some indication that positive feelings might transfer to others. In the second paragraph, we see the suggestion that positive affect can effect members of a group. "Happy feelings flit easily through social networks, so that one person's good fortune can brighten the day even for only distantly connected others." This sentence suggests transfer or a spread of positive feelings from one individual to other individuals. And that means that positive feelings can be seen as contagious, which is consistent with option C. Although it seems option C is a good answer, let's just check whether the fourth option might be better. The fourth option is that positive feelings are Ephemeral. Ephemeral means shortlived or temporary. Looking at the parts of the text where the author discusses positive feelings, which is mainly in the second paragraph, there is no clear implication that positive feelings are shortlived. There is an implicit implication that moods may vary by the moment at the end of paragraph three. And if positive feelings can change based merely on smiling and other people's feelings, as stated in paragraph two, then that might suggest that they are not highly stable. However, this inference is indirect and requires extra assumptions by the reader. So ephemeral is not as good of an answer as contagious, which directly captures a point made more explicitly in the passage. Because this question is asking you to identify an accurate paraphrase, or an accurate summary of an idea stated in the text, this is a foundations of comprehension question. And option C seems to be the best paraphrase of an idea from the text. Now let's try the second question. Suppose that economists do start using happiness instead of the gross national product as a measure of an economy's success. Information presented in the passage would predict which of the following: Statement 1. The transition will be frought with dificulty. Statement 2. The gross national product of the United States will appear to decrease. Statement 3. The economy of the United States will be seen as relatively less successful than today's. This question begins with the word suppose. That's a good clue that it's a reasoning beyond the text question. Asking you to assess how the new information, given to you in the question, will impact the ideas presented in the passage. The question asks you to imagine that economists do start to use happiness as a measure of the economy as suggested in the third paragraph of the passage. Then you're given three possible predictions to evaluate. The first prediction is that the transition will be frought with difficulty. So you should look through the passage to see if there is anything that suggests that switching to happiness as a measure might be difficult. Right after the author first mentions using happiness as a measure, in addition to the gross national product, the next sentence says, "Happiness is, of course, a slippery thing to measure or define." This implies that the transition to this metric will be difficult. So there is support for the first prediction. The second prediction, is that the gross national product of the United States will appear to decrease. Using happiness as a measure of success instead of the gross national product, doesn't suggest that anything would happen to the gross national product. The passage presents these as two separate metrics that might be used to evaluate economic success. So it doesn't appear that there is support for the second prediction. The third prediction, is that the economy of the United States will be seen as relatively less successful than today's. At first this prediction seems consistent with the suggestion that the US might be seen as unsucessful if economists switch to using a happiness measure. For example, in paragraph four, the author notes that when national happiness has been measured and compared to other countries, the US has not ranked particularly well. In fact, Americans rank only 23rd. The author also brings up the widespread use of antidepressants. However, the prediction is that the US economy would be seen as less successful using happiness than using the current standard. And the current standanrd is just based in the gross national product. So to know if the US economy would be seen as less successful, we would need to know where the US is ranked using just the gross national product. You are being asked what the passage itself predicts thus, you should not use any outside knowledge such as that the US ranks near the top in GNP to answer the question. Since there's no explicit mention of the US economy being ranked higher than 23rd in the passage, there is no support in the passage for the third prediction. Thus, the only prediction that is supported by the passage is the first one. And these types of questions which have both statements indicated by roman numerals, and response options indicated by letters, remember to complete the final step of deciding among the response options. Now let's try question three. What best represents the author's explanation for why Americans can be "so surpassingly positive in selfimage and stereotype without bein the world's happiest and bestoff people? A. American's posiivity is not a true reflection of their affect. B. Being welloff is not the same as being happy. C. Stereotypes tend to be unwarranted generalizations. D. Americans tend to have high rates of depression. Because this question is asking you to recognize an accurate paraphrase or summary of an idea stated in the text, this is a foundations of comprehension question. Here, we can see the quoted text is part of a sentence we marked at the start of the fifth paragraph. Following this first sentence, the author then provides us with an explanation that we need to summarize. In the second sentence, the author explicitly states, "Positivity is not so much their condition as it is part of their ideology." By this statement, the author is saying that positivity is not a true reflection of their condition. So this is consistent with option A. Before we decide that option A is the best answer, we need to look at the other options. Option B implies that the author discusses the difference between being welloff and being happy. But the author never does this. So it doesn't appear there's support in the passage for option B. And we can strike through that. Option C states that stereotypes tend to be unwarranted generalizations which suggests that most stereotypes can be seen in this way. The question itself tells you that the stereotype of Americans does not seem to match reality. But the author doesn't argue that most stereotypes are unwarranted generalizations. So it does not appear that option C is a good answer. Turning to option D, the passage does discuss high rates of the use of antidepressants in the US in paragraph four. But the purpose of including this information is to provide evidence for a lack of happiness among Americans. Not as part of an explination for how Americans can be "so surpassingly positive in selfimage and stereotype without being the world's happiest and bestoff people." So it doesn't appear that option D is a good answer either. And we can strike through that one. Option A best captures the authors message in response to the question. |
10 | Critical Analysis And Reasoning Skills Practice Questions | Critical Analysis And Reasoning Skills Tutorial | Worked example: Seeing color through Homer's eyes | The purpose of this video is to show you how I approach reading and answering questions on an example critical analysis and reasoning skills passage. The name of this passage is "Seeing Color Through Homer's Eyes." I'm going to read through the passage first. When I notice important sentences or signal words, I'll let you know that I'm highlighting them. Let's read the passage. "For someone used to contemporary academic writing, reading the chapter on color in William Gladstone's "Studies on Homer and the Homeric Age in 1858," comes as rather a shock, the shock of meeting an extraordinary mind. It is therefore all the more startling that Gladstone's 19th century tour de force comes to such a strange conclusion. Homer and his contemporaries perceived the world in something closer to black and white than to full technicolor." Hm, okay, so because the title of this passage is "Seeing Color Through Homer's Eyes," I'm going to guess that the last sentence is the topic sentence for this essay. And I'm hoping that the author's gonna tell us more about this strange conclusion. So for now, I'm gonna highlight that conclusion. Now let's go back to the passage. "No one would deny that there is a wide gulf between Homer's world and ours. In the millenia that separate us, empires have risen and fallen, religions and ideologies have come and gone, and science and technology have transformed our intellectual horizons in almost every aspect of daily life beyond all recognition. Surely one aspect that must have remained exactly the same since Homer's day, even since time in memorial, would be the rich colors of nature. The blue of sky and sea, the glowing red of dawn, the green of fresh leaves." Okay, so in this paragraph, the author tells us about a couple of reasons why things may have changed since Homer's time. But the last sentence proposes the assumption that colors in nature were exactly the same. This seems like a pretty important assumption, so I'm going to highlight it. Now let's go back to the passage. "Gladstone says things are not the same for many reasons, one, Homer uses the same word to denote colors, which according to us, are essentially different. For example, he describes as violet the sea, sheep, and iron. Two, Homer's similes are so rich with sensible imagery, we expect to find color a frequent and prominent ingredient, and yet his poppies have never so much as a hint of scarlet. Three, Gladstone notes, Homer uses black about 170 times, white 100 times, red 13, yellow 10, violet six times, and the other colors even less often. Four, Homer's color vocabulary is astonishingly small. There doesn't seem to be anything equivalent to our orange or pink in Homer's color palette. Most striking is the lack of any word that could be taken to mean blue." So the first sentence in this paragraph is in direct response to the important assumption stated at the end of the third paragraph. The end of the last paragraph said, "surely the colors of nature have remained the same." Whereas the start of this paragraph says Gladstone says things are not the same. That seems pretty important, so I'm highlighting that point. Then what follows are Gladstone's many reasons, the evidence for this suggestion comes from Gladstone's analysis of Homer's language. These many reasons are even numbered for us. Okay, now I'm gonna continue reading to see what comes next. "What is more, Gladstone proves that the oddities in Homer's 'Iliad' and 'Odyssey' could not have stemmed from any problems peculiar to Homer. Violet colored hair was used by Pindar in his poems. Gladstone is well aware of the utter weirdness of his thesis. Nothing less than universal colorblindness among the ancient Greeks. So he tries to make it more palatable by evoking an evolutionary explanation for how sensitivity to colors could have increased over the generations. The perception of color, he says, seems natural to us only because humankind as a whole has undergone a progressive education of the eye over the last millenia. The eye's ability to perceive and appreciate differences in color, he suggests, can improve with practice and these acquired improvements are then passed on to offspring." In these paragraphs, the author clarifies that Gladstone's thesis is not really about something peculiar to Homer, but something common to all ancient Greeks. He gives an example from Pindar to make his point. Then he gives Gladstone's explanation that the perception of color required practice and evolution. Both of these seem like important points, so I'll highlight both the first and last sentence of the fifth paragraph. Now let's go back to the passage. "But why, one may well ask, should this progressive refinement of color vision not have started much earlier than the Homeric period? Gladstone's theory is that the appreciation of color as a property independent of a particular material develops only with the capacity to manipulate colors artificially. And that capacity, he notes, barely existed in Homer's day. The art of dying was in its infancy. Cultivation of flowers was not practiced, and almost all of the brightly colored objects we take for granted were entirely absent. Other than the ocean, people in Homer's day may have gone through life without ever setting their eyes on a single blue object. Blue eyes, Gladstone explains, were in short supply. Blue dyes, which are very difficult to manufacture, were practically unknown. And natural flowers that are truly blue are rare." So this paragraph gives us another part of Gladstone's theory that the refinement of color perception did not become important until people had developed the ability to manipulate color through dying. I'm highlighting the sentence about that point. Now let's finish the passage. "Gladstone's analysis was brilliant, but completely off course. Indeed, philologists, anthropologists, and even natural scientists would need decades to free themselves from the error of underestimating the power of culture. Adopted from G. Deutscher, "Through the Language Glass Why the World Looks Different in Other Languages, copyright 2010, Metropolitan Books." So the final section gives you the author's perspective on Gladstone's theory, namely, it seems he does not believe it, as suggested by the phrase, "completely off course." I'll highlight that phrase in case a question asks me about the author's perspective. The final sentence also gives more detail on how the author thinks Gladstone's theory is wrong, but then the passage ends without providing an argument supporting an alternative view. Now let's look at the first question. It can be inferred from the passage that the author believes which of the following about contemporary academic writing? A) Academic papers are typically not especially brilliant. B) Academics seldom address color perception in their papers. C) Academics often reach very strange conclusions in their papers. D) Academic papers are usually outdated soon after they are written. This question is asking us to summarize or paraphrase the author's view of contemporary academic writing. Because it's asking you to summarize or paraphrase a point or idea from the passage, this is a foundations of comprehension question. Skimming the passage, we can see that the author mentions contemporary academic writing in the first sentence. The author begins the passage by saying that, "for someone used to contemporary academic writing, reading the chapter on color in William Gladstone's "Studies on Homer and the Homeric Age in 1858," comes as rather a shock, the shock of meeting an extraordinary mind." If someone used to reading contemporary academic writing is shocked at reading something extraordinary, then the implication is that academic papers are not typically especially brilliant. This is consistent with option A, however, before we select that answer, let's just check that none of the other answers are better. The passage does not discuss the popularity of color perception as a topic in contemporary academic writing, so it does not appear that option B is a good answer, and we can strike through that. The author does refer to the conclusion that Gladstone reaches as being strange, but does not suggest that this is true for many papers or that this happens often, so it does not seem that option C is a good answer, and we can rule that one out too. Finally, the author does not discuss the idea that papers become quickly outdated anywhere in the passage. And in fact, the last sentence suggests that Gladstone's ideas, even though incorrect, had a strong influence on other academics for decades. So we can rule out option D too. Option A is the best summary of the author's beliefs about contemporary academic writing. Question two, it has been suggested that "The Iliad" and "The Odyssey" were a patchwork of a great number of popular ballads woven together from different poets, rather than a single work by a poet named Homer. If true, how would this affect the opinions expressed in the passage? A) It would strengthen Gladstone's basic thesis. B) It would weaken Gladstone's basic thesis. C) It would require a modification of Gladstone's basic thesis. D) It would not affect Gladstone's basic thesis. The last sentence in the question stem starts with "if true," which tells you that you're being asked to think about an imaginary or hypothetical situation. This means this is a reasoning beyond the text question where you need to extrapolate the ideas in the passage to a new situation or assess how the new information would impact the arguments presented in the passage. So first we need to remember what Gladstone's main thesis was. We can return to the sentence we highlighted at the start of the fifth paragraph that states that Gladstone's thesis was "nothing less than universal colorblindness among the ancient Greeks." If "The Iliad" and "The Odyssey" were actually works composed by a great number of writers, then it would help to show that both Homer and other ancient Greeks used a restricted range of colors in their writings, rather than just a single individual like Homer or two individuals like Homer and Pindar. Thus, this would greatly strengthen Gladstone's basic thesis because it would suggest that colorblindness was not just peculiar to Homer, but was more universal. This reasoning supports option A, and is inconsistent with all of the other options. If the new information strengthens Gladstone's main thesis, then it would affect it, and would not weaken it, nor would it require it to be modified. Only if Gladstone were making an argument specific to Homer would the new information weaken his thesis. Thus, none of the other options are correct. Question three, Gladstone would predict which of the following about the children of an interior decorator, who easily distinguishes among scarlet, burgundy, and fuchsia? A) The children would be able to easily distinguish various versions of red. B) The children would be drawn more to objects in various versions of red than to those of any other color. C) The children would seldom bother mentioning what are to them obvious differences among various versions of red. D) The children would need practice distinguishing among various versions of red for years before achieving proficiency. In this question we are given a new situation not mentioned in the text, and are asked to make a prediction about it. Because we're given a new situation, this is a reasoning beyond the text question. To answer it, you need to either apply or extrapolate the ideas in the passage to the new situation. This question is asking us to make a prediction about color perception. Skimping back over the passage, this question seems most related to the sections where the author unpacked Gladstone's explanation of why the Greeks may have been colorblind but people today are not. In the sentence we highlighted at the end of the fifth paragraph, the author says that Gladstone suggests that the eye's ability to perceive and appreciate differences in color can improve with practice. And these acquired improvements are then passed on to offspring. So this idea suggests that the children of an interior decorator would be similarly able to distinguish various versions of red, which is consistent with option A. Before we select that as the best option, let's check out the other responses. The passage doesn't seem to talk about preferences for colors or attraction to colors at all. So there doesn't seem to be support for the prediction that these children would be attracted to objects of a certain color, which rules out option B. In addition, there's no suggestion that if you have the ability to distinguish among colors, that you wouldn't mention the differences that you see. In fact, the opposite is likely to be true based on Gladstone's analysis. He equates the use of color names with the limited perception of colors. So we can rule out option C. Although Gladstone's theory does suggest that improvements can be acquired through training, he also suggests that these improvements can be passed on to offspring. The theory doesn't suggest that children who inherit these improvements would also need to practice, so option D is not a prediction that follows directly from the text. Option A is the prediction that best follows from the passage. Question four, Homer's sky is starry or broad or great or iron or violet, but it is never blue, how does this affect the opinions expressed in the passage? Option one, it supports Gladstone's claim regarding Homer's use of color. Option two, it extends Gladstone's claim regarding Homer's focus on nature. Option three, it challenges Gladstone's claim regarding Homer's penchant for strange imagery. In this question, you're given new information beyond what is given in the passage. This means this is a reasoning beyond the text question, where you need to assess how the new information would impact the ideas presented in the passage. To evaluate the first statement, it supports Gladstone's claim regarding Homer's use of color. We need to remind ourselves of Gladstone's claim. Gladstone's thesis is that Homer's failure to use color was due to "nothing less than universal colorblindness among the ancient Greeks." The new information in this passage is telling us that Homer never uses the word blue to describe the sky. Later in the passage, there are a number of sentences discussing the color blue. "Other than the ocean, people in Homer's day may have gone through life without ever setting their eyes on a single blue object. Blue eyes, Gladstone explains, were in short supply. Blue dyes, which are very difficult to manufacture, were practically unknown and natural flowers that are truly blue are rare. The new fact that Homer never uses blue to describe the sky seems consistent with both this discussion of the color blue as well as the main thesis about the Greeks. So statement one is a good option. The new information supports Gladstone's claims about Homer's use of color. Statement two asks you whether the new information extends Gladstone's claim about Homer's focus on nature. Looking back through the passage, there's no direct discussion about Homer focusing on nature. The passage does give some examples of Homer writing about things in nature, and you could argue that Gladstone's claim is that although Homer is trying to describe nature, he fails to mention most colors when he does. Thus, failing to mention blue when describing the sky could be seen as consistent with the way Gladstone portrays Homer as describing nature. However, since Gladstone does not actually make a claim about nature being the focus of Homer's writing, statement two is not as defensible as a good answer as statement one. Statement three asks you whether the new information challenges Gladstone's claim that Homer's similes are rich with strange imagery. Looking through the passage, we see that Gladstone referred to Homer's writing as being rich with sensible imagery, not strange imagery. Since Gladstone does not make this claim, the new information cannot support it and statement three is not a good response. In these types of questions which have both statements indicated by Roman numerals and response options indicated by letters, remember to complete the final step of deciding among the response options. In this case, our four options are A, statement one only, B, statement two only, C, statements one and three, D, statements two and three. Statement one is clearly correct. While a weaker argument could be made for statement two, but there's no answer option that includes both statements one and two. Thus, we should select option A. |
11 | Critical Analysis And Reasoning Skills Practice Questions | Critical Analysis And Reasoning Skills Tutorial | Worked example: Physical education in the UK | The purpose of this video is to give you an idea how to approach reading and answering questions on the Critical Analysis and Reasoning Skills section. The name of this example passage is physical education in the UK. I'm going to read through the passage first. When I notice important sentences or signal words, I'll let you know that I'm highlighting them. In the United Kingdom, physical education, PE, is compulsory in state schools until students reach the age of 16. That is, sports are compulsory for as long as formal education is mandated by law. Because there are many children who don't want to participate in PE classes, I believe that students should be allowed a choice. If their parents agree, why should they be forced to jump on a trampoline or do calisthenics? PE class is different from other classes because it involves what one does with one's body. We acknowledge the rate of individuals to control their own bodies to determine whether and when they have an operation, to determine where they go and what they do. Why is this any different? So in the third sentence, the author comes right out and tells us their position on this issue. It's pretty clear that this text is going to be about why students should not be forced to take PE. The author then goes on to make the argument that because PE involves one's body, that it's different than other classes and it violates our rights to not have control over our own bodies. This point seems pretty important, so I'm going to highlight it so I can find this argument later. I'm also going to highlight the third sentence where the author told us his opinion. It is a red herring to say that PE makes any serious difference to people's health. There are more effective ways of ensuring a healthy population than pushing children to run laps around a freezing sports field once a week. For example, schools could be addressing the poor diets young people have today and encourage them to walk or bicycle to school rather than rely on the car. Furthermore, sports are a waste of school time and resources. One or two PE lessons a week makes very little difference to an individual's health, but they make a huge difference in the school's budget. Mandatory PE requires a whole extra department in schools, wasting a great deal of money and time that could be better spent on academics. It also requires schools to be surrounded by a large amount of land for playing fields, making it prohibitively expensive to build new schools in urban areas. Given the average current pupilteacher ratios, the quality of teaching in PE classes is necessarily low, and the classes may even be dangerous to students who are not properly supervised. Our children are burdened enough in schools already, especially at the older end of the system, with multiple examinations. PE simply adds needlessly to this hectic schedule. In this paragraph, the author expands the argument. In addition to dismissing the idea that PE should be required because it's good for your health, the author goes even further to suggest that sports should be eliminated from schools entirely because they're a waste of resources. Then the author questions the quality of PE classes and argues that they make student schedules more hectic. I'm going to go back through this paragraph and highlight the sections for each of these points. Many people argue that playing team sports builds character, encourages students to work with others, teaches children how to win and lose with good grace, and builds strong school spirit through competition with other institutions. It is often, they say, the experience of playing on a team together which builds the strongest friendships at school, friendships which endure for years afterwards. Many say the same benefits derive from the common endurance of prison. Injuries sustained through the school sport and the psychological trauma of being bullied for sporting ineptitude can mark people for years after they have left school. On that note, in an increasingly litigious age, a compulsory rather than voluntary sports program is a liability. More and more schools are avoiding team games such as rugby, soccer, hockey, and football due to the realistic fear of lawsuits. Teamwork can be better developed through music, drama, and community projects without the need to encourage an ultracompetitive ethos. Here, the author refutes a likely argument in favor of sports by mentioning several negative consequences including injury, ridicule, and lawsuits. I'm going to highlight each of these. By comparing being on a team to being in prison, and asserting that teamwork is better developed in other contexts, it's pretty clear that the author has a negative view of sports. I'm going to highlight these clues as well. As for the argument that without compulsory PE many members of society wouldn't find out if they had a talent for a sport, or even if they enjoyed it, students can discover this aptitude outside of school without also discovering the bullying and humiliation that comes with PE classes more than with other lessons. The aim of compulsory PE isn't being fulfilled at present in any case, as sick notes are produced with alarming regularity by parents complicit in their children's wish to avoid it. Greater efforts to enforce it will only result in more deceit, children missing school for the entire day or in the most extreme cases, children being withdrawn from state education. In this final paragraph, the author adds a few more points. The author suggests that students can discover their talents outside school, and reiterates the benefits of removing the opportunity for humiliation. Finally, the passage ends by suggesting that many people are already finding ways to avoid PE, and warns that efforts to enforce participation could result in avoidance of school altogether. Adapted from A. Dean, Physical Education Compulsory, Creative Comments, copyright 2011, Creative Comments. Question one, what is the function of the statement in the first paragraph that PE class is different from other classes? A, it's part of an argument why PE classes should be required, B, it's part of an argument that PE classes improve people's health, C, it explains why students should only be exempt from PE with parental permission, D, it explains why students should have a choice about whether to take PE while not having a choice about taking other compulsory classes. This question is asking you about the role of a particular statement or idea in the author's argument. Because it's asking about the author's reasoning, it's a reasoning within the text question. If we return to the first paragraph, we can see that this phrase, PE class is different from other classes because it involves what one does with one's body, comes after the sentence where the author states I believe that students should be allowed a choice. So this statement forms part of the author's argument why students should have a choice, which is most consistent with option D. The author explicitly rejects that PE should be required in this first paragraph, so option A is not a good answer. In the second paragraph, the author dismisses the idea that PE is a good way to improve people's health. This is inconsistent with option B, which suggests the opposite. Further, the observation that PE class is different from other classes does not appear as part of this discussion about health, which means option B is not a good answer for multiple reasons. Although the author does mention the idea of parental permission in paragraph one, the idea that PE class is different from other classes is not directly linked to the idea about parental permission, suggesting that option C is not as good an answer. The option that best describes the role of this statement in the author's reasoning is option D. Question two, which of the following assumptions is made by the author in relation to the argument about student's hectic schedules? A, PE tends not to have a final examination, B, PE tends not to have a heavy homework burden, C, compulsory PE, if eliminated, would not be replaced by another compulsory course, D, it's unfair to require students in the higher grades to prepare for multiple examinations. Looking back at our highlighting, we can see that the reference to hectic schedules occurs at the end of the second paragraph. The author describes the busy schedules of students and argues for the elimination of PE to help alleviate that burden. This would only be true if PE was not replaced by another compulsory course with a similar amount of work involved. Option C, the fact that PE tends not to have a final examination, as stated in option A, and does not have a heavy homework burden, as stated in option B, suggests that PE might be relatively less likely to cause additional stress for a student relative to another class. This seems the opposite of the author's argument, and although these might seem like obvious facts about PE, the author doesn't imply them, nor do they need to be assumed to make the author's point. Although he author implies that students at the older end experience the most burden, there's nothing in the text that implies that the author believes this is unfair. So option D is also not a good option. Option C is the assumption that would most affect the author's reasoning if it were not true. Question three, assume it's true that students are more likely to obtain specialist coaching at sports clubs outside of school than in school. How would this information be relevant to the passage? A, it would restate an objection to compulsory physical education classes, B, it would support a point about discovering sports aptitude made in rebuttal, C, it would directly challenge one of the author's claims, D, it would contradict one of the author's examples. This question starts with the phrase, Assume as true, which is a good clue that this is a reasoning beyond the text question. You're given a new situation to think about, and you're asked to assess how the new information impacts the ideas presented in the passage. Skimming through the passage, we find that this new information seems most relevant to ideas in the fourth paragraph where the author talks about sports clubs outside school. The author writes, As for the argument that without compulsory PE, many members of society wouldn't find out that they had a talent for a sport, or even that they enjoyed it. Students can discover this aptitude outside of school without also discovering the bullying and humiliation that comes with PE classes more than with other lessons. If it is true that students are obtaining specialist coaching at sports clubs outside of school more often than in school, then it would further support this point. The author never claims that outside clubs do not have specialist coaches, nor are there any examples of this in the passage. So this new information would not directly challenge or contradict anything we read. In the third paragraph, the author mentions that the quality of teaching in PE classes is low. The new information is consistent with this idea because it suggests that more specialized coaching, which can be supposed to be higher quality, is available outside of school. But this new information is not just a simple restatement of the idea in paragraph three. It adds new information about the quality of coaching. Another reason why option A is not correct is because the point about low quality in schools is made as part of an objection to any sports in school. Option A is talking about restating an objection against compulsory PE. So for a couple of reasons, option B is the better answer. Question four, the author's central theme for the whole passage is A, opposing formal educational mandates, B, describing the consequences of making PE compulsory, C, presenting reasons why PE should not be compulsory, D, advocating that PE be abolished in UK state schools. This question is asking about the main theme. This means it's a foundations of comprehension question. The author argues in the third sentence that students should be allowed a choice. The author describes the rights of individuals to control their own bodies in other settings in order to argue for an exception from mandates for PE classes in particular. The author's main point is not opposing educational mandates or compulsory education in general, but only in the instance of PE. So option A is not a good answer. Although the author describes a number of possible negative consequences of PE, this represents only part of the passage. So it does not appear that option B will be the best answer. The author does discuss the reasons why sports should be eliminated from schools, but again, this is only part of the passage. So option D is probably not the best answer. Option C provides the most general theme for the passage. The idea that PE should not be compulsory is related to all of the claims made in the passage. |
12 | Critical Analysis And Reasoning Skills Practice Questions | Critical Analysis And Reasoning Skills Tutorial | Worked example: The honest truth about dishonesty | The purpose of this video is to show you how I'd approach reading and answering questions on an example Critical Analysis and Reasoning Skills passage. The name of this passage is The Honest Truth About Dishonesty. I'm gonna read through the passage first. When I notice important sentences or signal words, I'll let you know that I'm highlighting them. Here's the passage. "Although business people deserve more respect for their honesty than they receive, a common complain is that they take advantage of consumers through dishonest advertising. Instead of providing useful information for making rational choices, Advertisements often appeal to consumer's emotions, to persuade them to buy products regardless of need. This complaint is true and obvious to all but the most naive people. Advertisements are designed to convince consumers to favor one product over others, and presenting solely unbiased and unemotional information would seldom be the best way to accomplish this goal. Thoughtful people recognize that politicians advertise themselves and their policy recommendations in similarly biased and emotional ways. The question is not whether business people or politicians have the strongest moral commitment to truthfulness in advertising. Both groups will deviate from honest practices when they expect that the benefits of doing so will exceed the costs. The important question is, who can most easily mislead their customers with emotional statements, unrealistic promises and biased information. Business people, or politicians." So that seems pretty clear that the author is trying to tell us that the last sentence is going to be important cause they say, "The important question is.." I'm gonna highlight that sentence so I can find it later. Now I'm gonna go back to the passage to read some more. "People are less likely to be swayed by dishonesty and emotion when responding to business ads than when responding to political ads, for two reasons. First, business people are attempting to persuade people who are usually spending their own money. Politicians, are trying to persuade people who are deciding how they want to spend other people's money. The motivation to minimize mistakes by carefully considering claims about costs and benefits, before a decision is made, and by evaluating those claims in light of post decision experience is greater when one is bearing all of the cost of the decision than when others are bearing most of the cost." This first sentence seems important cause it gives us a thesis statement. It tells us what the author is going to argue for. I'm going to highlight that claim. Then I notice in the first sentence, the phrase, "for two reasons." That's also an important signal. I'm gonna highlight that. In the next sentence, the author then uses the word "first" and goes on to tell us the first of those reasons. Reading through it looks like the rest of the paragraph is still all about that first point. I don't see a second reason in there so hopefully that's still coming. I'm gonna go back to the passage now to see what happens next. "The second reason why misleading claims are less effective in promoting commercial products, than in promoting political products is because the choices that consumers of commercial products make have more decisive effects on outcomes than do the choices of consumers of political products. When people purchase the product in the market place they get the product they choose and they get it because they chose it. The probability that a voter's choice will be decisive is increasingly small in State and Federal Elections and seldom greater than a fraction of 1% in most local elections. Given such a low probability of any one person's vote determining the outcome of the election, voter's have little motivation to be concerned about the accuracy of political claims being made." Great! So this paragraph gives us the second of the two reasons that we were looking for. I'm highlighting the phrase "The second reason" so I can find it later. Now I'm gonna go back to the test. "One might think that professors would be more honest than both business people and politicians when promoting their product's value, that is, in their Teaching and Research. Unlike politicians professors try to sell their products to customers who can decisively accept or reject them without being directly affected by how many others make different choices. However, many undergraduate students are glaringly indifferent to what professors have to say, so professors have more latitude than business people to benefit from exaggerated or deplecious claims." The first sentence to this paragraph uses a hypothetical statement "One might think.." The author is using that to introduce a new claim. The author is also shifting from discussing just business people and politicians to considering a third group of people, professors. So for both of those reasons I'm gonna mark this sentence cause it gives us another main claim, and it marks a transition in the topic that might be important later. Also, the word "so" in the last sentence shows us a conclusion that the author is trying to make. The conclusion asserts that professors have more to gain from dishonesty than business people so I'm going to mark that too Now I'm going back to finish the passage. " Professors have to be more restrained when publishing than when teaching, because other professors will evaluate the truth of their published claims. It is true that academic promotions may be earned and scholarly reputations enhanced by exposing the errors in published work. However, professors are often less concerned with the truthfulness of articles written by other professors than one might think. Professors anxious to get their own articles and books published are often less interested in whether the publications they cite are correct, than in whether the publications are accepted as correct by academics with views similar to their own: the people most likely to decide whether the books and articles will be published and cited." We finally see the passage for this citation at the end adapted from D. Lee, "Why Businessmen Are More Honest Than Preachers, Politicians and Professors." Copyright 2010, Independent Review Here the title makes it pretty clear what the author's message is gonna be. I'm gonna highlight this title to help me later as well. Now let's look at the first question for this passage. "The author implies which of the following about business people and politicians? A. Neither are very thoughtful people. B. Neither have a strong moral commitment to truthfulness. C. Both have biased views about their customers and constituents respectively. D. Both are more concerned about advertising themselves than their products respectively." This question is asking you which of these ideas the author is trying to convey in the passage. Because the question asks you which of these ideas the author "Implies" instead of "says", this is a clue that you're not looking for a direct quote, but rather you're looking for a part of the text that could be paraphrased or summarized as one of these statements. Because you're being asked to identify a paraphrase or summary from the passage, this is a Foundations of Comprehension question. Looking through the first paragraph, the first sentence tells us that the section will be about business people. Let's reread that sentence. "Although business people deserve more respect for their honesty than they receive, a common complaint is that they take advantage of consumers through dishonest advertising." The first sentence introduces business people as the subject and it suggests that a common complaint about business people is their dishonesty. However, in the first part, the author tempers this complaint by saying, "business people deserve more respect for their honesty than they receive.." This clause suggests that the author thinks people are too extreme in their beliefs that business people are dishonest The next sentence provides support for that idea that advertising is dishonest. Let's reread that. "Instead of providing useful information for making rational choices, advertisements often appeal to consumer's emotions to persuade them to buy products regardless of need." And the third sentence shows the author's explicit endorsement of the idea that advertisements are dishonest. If we read that again, we can see "this complaint is true and obvious to all but the most naive people." The phrase "obvious to all but the most naive people" shows you a little bit of the author's attitude. The language is meant to be insulting as it tells you that you're naive if you don't agree with the author. The final sentence in this first paragraph continues the argument that advertisements are dishonest. In this sentence the author adds the idea that advertisements are actually designed to be dishonest. Let's read that sentence again. "Advertisements are designed to convince consumers to favor one product over others, and presenting solely unbiased and unemotional infromation would seldom be the best way to accomplish this goal." Looking back at our four response options, it doesn't appear that in this first paragraph there are any statements related to the lack of thoughtfulness in business people, so it doesn't appear that we have any evidence for option A yet. We also don't see any evidence that business people are concerned with advertising themselves, so it doesn't look like we have any evidence for option D yet. The paragraph does talk about bias, but this is in relation to advertising not in relation to business people's views of their own customers, so it doesn't look like we have any evidence for option C. At the same time, there is some information about dishonesty in advertising, that suggests that the author might question whether business people have a moral commitment to truthfulness. So far, we have a little evidence in favor of option D. Let's go back to the passage now and see what we can find in the second paragraph. In the first sentence of the second paragraph, the author starts to talk about politicians. You also find the word thoughtful as well as the idea that politicians advertise themselves. Let's reread that sentence. "Thoughtful people recognize that politicians advertise themselves and their policy recommendations in similarly biased and emotional ways." When you read closely you find that "thoughtful" is used to describe people who recognize that politicians advertise themselves. It's not used to describe either business people or politicians. This sentence doesn't imply that either business people or politicians are not thoughtful, so this sentence doesn't provide any support for option A. Further this sentence also talks about the fact that politicians advertise themselves, but does not mention anything about business people sharing that goal, so this sentence doesn't provide any support for option D. The next two sentences explicitly discuss the moral commitment to truthfulness among business people and politicians. Let's read this sentence again. "The question is not whether business people or politicians have the strongest moral commitment to truthfulness in advertising. Both groups will deviate from honest practices when they expect that the benefits of doing so will exceed the costs." Thus, because both business people and politicians will deviate from honest practices when it's in their own interests, these two sentences imply that the author believes that neither have a strong moral commitment to truthfulness, which is consistent with option B. Skimming through the remainder of the passage, you can confirm that there's no language that suggests that business people and politicians are not thoughtful, so we can finally reject option A. The passage also doesn't discuss whether business people or politicians are biased in their views of their customers or their constituents, and that allows us to reject option C. Finally, while politicians may be more likely to advertise themselves, the passage describes business people as generally trying to advertise their products, which allows you to reject option D. Now let's try a second question. "Which of the following assumptions is most central to the author's argument? Option A. Most products are designed to appeal to naive and emotional consumers. Option B. Products are more likely to be purchased when they are advertised than when they are not. Option C. If business people manufactured only products that people need there would be few products on the market. Option D. If products were evaluated according to objective information about them, people would often not prefer one over the other. Looking at these response options, this question is asking you to think more deeply about the author's reasoning about the relation between products and advertising. In reviewing the passage, you can see that topics of products and advertising are the focus of the first paragraph. So that's a good place to start to try to see which of these assumptions the author is making. When you're being asked about the parts of an author's argument, including their claims, evidence or assumptions that's a good clue that you're being asked to think about the author's reasoning. These types of questions fall under the "Reasoning within the text" category. Looking at the first paragraph, in the second sentence the author writes' "Instead of providing useful information for making rational choices, advertisements often appeal to consumer's emotions to persuade them to buy a product regardless of need. In this sentence the author suggests that advertisements need to appeal to consumer's emotions. Contrary to option C, this doesn't assume that most products on the market are not needed by anyone, only that some of the people who buy some products do not need them. In the fourth sentence the author talks about how non emotional and objective information would not be effective in getting people to choose the advertised product over others. "Advertisements are designed to convince consumers to favor one product over others, and presenting solely unbiased and unemotional information would seldom be the best way to accomplish this goal." This implies that factual information is not sufficient as a basis for consumers to form a preference, and thus this points to D as a valid answer. Combined with the sentence above, the implication is that merely advertising a product will not lead to more purchases. This is contrary to the assumption in option B. Instead, only emotional advertisements would have this effect. Thus, it seems the author is assuming D but not B. In addition, this fourth sentence does not imply that the author believes that products need to be designed to appeal to emotions, rather it refers to advertisements for the products being designed to appeal to emotions, so this implication is not consistent with what is claimed in option A. Finally, the author's reference to naive people in the third sentence, is a reference toward anyone who disagrees with the author, not towards product consumers as stated in option A. In some, all the options other than D require misinterpretation of statements made by the author, or require going beyond what the author is saying. Thus only option D is something that the author is assuming in his argument. Let's do a third question. "Suppose a politician is reelected despite lying about his voting record, the passage suggests which of the following explanations? A. The politician made many contradictory statements during his or her campaign. B. For the second election were significantly different than for the first. C. Voters did not compare the politicians behavior while in office with statements made during his or her campaign. D. There was no consensus among voters regarding the cost in benefits of a second term in office for that politician." This question is asking about a new situation that was not mentioned in the text. The word "suppose" is a good clue that you're going to be asked to reason about a new situation. When a question introduces a new situation or asks you to apply or extrapolate ideas to a new context, then you're being asked a " Reasoning beyond the text" question. The topic of this question is about politicians, and how people might react to dishonesty. So you know that the information that might help you to answer this question will come later in the passage since the first paragraph is only about business people. The second paragraph starts to be about politicians. As we already noted while reading the text, the second paragraph then ends with an important question. Let's reread it. "The important question is, who can most easily mislead their customers with emotional statements, unrealistic promises and biased information, business people or politicians?" This alerts you that a main focus of the author's argument is going to be people's reactions to the dishonesty of business people or politicians. Because you're looking for information on how people might react to dishonesty, it seems like the part of the passage that follows this, the third paragraph is gonna be important. The first sentence of the third paragraph tells you that the author is going to argue that politicians may be more likely to get away with being dishonest. Let's reread that sentence. "People are less likely to be swayed by dishonesty and emotion when responding to business ads, than when responding to political ads for two reasons.." Then the remainder of that paragraph outlines the first reason why politicians may be more likely to get away with being dishonest. Let's reread that. "First, business people are attempting to persuade people who are usually spending their own money. Politicians are trying to persuade people who are deciding how they want to spend other peoples money. The motivation to minimize mistakes by carefully considering claims about costs and benefits before a decision is made, and by evaluating those claims in light of postdecision experience is greater when one is bearing all of the cost of the decision, than when others are bearing most of the cost. " So the author is arguing that individuals are more willing to carefully evaluate the claims of a business person over the claims of a politician. The author gives their second reason in paragraph Four. Let's reread that paragraph too. "The second reason why misleading claims are less effective in promoting commercial products, than in promoting political products is because the choices that consumers of commercial products make have more decisive outcomes than do the choices of consumers of political products. When people purchase a product in the market place, they get the product they choose and they get it because they chose it. The probability that a voter's choice will be decisive is increasingly small in State and Federal elections and seldom greater than a fraction of 1% in most local elections. Given such a low probability of any one person's vote determining the outcome of the election, voters have little motivation to be concerned about the accuracy of political claims being made." So, if a politician is able to get reelected even though they lied about their voting record, then the passage is suggesting that the voters were generally not as motivated to evaluate the politician's claims over business people's claims. This is most consistent with option C where comparison of behavior against statements can be seen as a way of assessing the honesty of the politician. Voter's do not compare the politician's behavior while in office with statements made during his or her campaign. The paragraphs we just read don't talk about the presence of contradictory statements as reasons why people are less likely to be affected by the dishonesty of political ads, so there's no evidence consistent with option A. These paragraphs also do not discuss changes in the cohorts of voters. It is possible that a new cohort of voters elected the politician in the second term, but the important thing to remember when answering these questions is that this alternative was not discussed by the passage, so we don't have any evidence in favor of option B. The notion of costs and benefits that is mentioned in that third paragraph is part of the careful evaluation process that people usually only engage in for business ads because the consumer bears all of the cost of a mistake. The author does not assume that voters are likely to perform a similar costbenefit analysis, so whether or not there's a consensus and a costbenefit analysis would be irrelevant. Also the paragraph does not discuss a lack of consensus among people on costbenefit analysis as a reason why people might be less likely to be affected by dishonesty in any context, so we don't see any evidence to support option D. Let's look at one final question for this passage. "The author most likely mentions Probability in his discussion of voting behavior as reasoning for which of the following in paragraph Four? Statement One. To explain low voter turn out in state and federal elections Statement Two. To explain the prevalence of politicians' dishonesty. Statement Three. To explain why voters do not carefully consider political claims." This question's a bit tricky because it gives us three statements that we need to choose between, and then it gives us a bunch of response options. Let's return to the response options after we've thought about the statements. The main question is, "Why does the author discuss probability as part of his argument?" You're being asked which explanation in the text is supported by the discussion of probability. This means you're being asked to think more deeply about the author's reasoning, and these types of questions fall under the "Reasoning within the text" category. Since the question tells you that the reasoning you're being asked about is in Paragraph Four, that's a good place to start. Skimming through the paragraph, you can see the first mention of probability is in the third sentence. Let's read that sentence. "The probability that a voter's choice will be decisive is increasingly small in state and federal elections, and seldom greater than a fraction of 1% in most local elections." Then the fourth sentence helps to tell you why the author thinks this point is important. Let's reread that sentence. "Given such a low probability of any one person's vote determining the outcome of the election, voter's will have little motivation to be concerned about the accuracy of the political claims being made." So the second part of this sentence tells you the point that the author is trying to make by mentioning probability. The author is saying that the individual voter has a very small impact on the outcome of the election, and therefore each voter has only a small stake in the overall decision. The lack of a sense of ownership is use to explain why voters do not carefully consider political claims, and this is consistent with statement Three. The author explicitly tells you in the first sentence that the thing that is going to be explained in the fourth paragraph is why misleading claims can be effective in political contexts. Let's reread that sentence. "The second reason why misleading claims are less effective in promoting commercial products than in promoting political products is..." So the author is not trying to explain low voter turn out, and you can rule out statement One. The explanation being developed by the author is why people react to dishonesty differently in political and business contexts. Although the dishonesty of politicians is implied by the passage, the discussion of probability is not included as part of an explanation about why politicians are dishonest. So statement two is not a good response. It may be tempting to infer that the author is trying to argue that the effectiveness of dishonesty in political advertising makes it more prevalent in politics, but the author never makes that connection. In fact, the author explicitly rejects the value of making comparison about the prevalence of dishonesty in paragraph Two. If we could reread that sentence it says, "The question is not whether business people or politicians have the strongest moral commitment to truthfulness, both groups will deviate." So now that we have reviewed the statements, remember there's a final step in answering these kinds of questions that have those statements indicated by Roman numerals, and response options indicated by letters. Now that you have determined that statement Three is the only one that accurately describes the relation between the discussion of probability and the explanation being given by the author, you need to pick the correct response option, in this case, the answer is B. |
13 | Biomolecules | Amino Acids And Proteins1 | Central dogma of molecular biology | So what exactly is the central dogma of molecular biology? Well, really it could just be called the central dogma of all of life because it explains how you and I take this conglomeration of genetic information from each of our parents, and how this information gets transferred into generating a fullblown human being, like you and me. So some very clever scientists, Francis Crick and James Watson, or "Watson and Crick," as they're often referred to as, are credited with discovering this dogma, which they say deals with the detailed residuebyresidue transfer of sequential information. Or, as Marshall Nirenberg, who won the Nobel Prize in Physiology and Medicine, once said, "DNA makes RNA makes protein." And I think this simple explanation really just says it all. So let's explore this concept a little bit further. So we have three major players here. DNA and RNA are nucleic acids, which are made up of nucleotides, and proteins are made up of amino acids. And the information starts at the most basic level stored as DNA, which can then be restored as DNA when DNA copies itself in a process called "replication." Then DNA can be copied into RNA in a process called "transcription." And then finally you can use the information in RNA to synthesize a protein in a process called "translation." Now since DNA, RNA, and protein are linear polymers, this means that each individual unit, or monomer, is only attached to, at most, two other units. So say we have a monomer, which is just one unit. They are connected in a series like this, which makes it a linear polymer, and this is the same for DNA if each of these is a deoxyribonucleic acid, for RNA if it's a ribonucleic acid, or a protein, which are just amino acids all connected in a linear polymer. So what does this mean? This means that the specific sequence of each of these monomers effectively encodes information, and that that transfer of information is faithfully preserved from DNA to RNA to protein. Each polymer sequence is used as a template for the synthesis of the next polymer. And you could go into any step in this sequence and determine what the corresponding polymers would look like. So in other words, you could take DNA and obviously figure out what the corresponding RNA would look like, and then what the corresponding protein would look like. So this whole process is the central dogma. It can sometimes be a little bit tricky to keep all of these terms straight, so I'll try to break it down a little bit for how I like to remember them. For DNA, I think it's pretty easy. When you go from DNA, and DNA makes a copy of itself, it's called replication because DNA is just replicating itself. It's making the same copy of itself. Transcription and translation, on the other hand, it's kind of easy to get these two terms mixed up. One of them obviously is talking about DNA to RNA, whereas the other one is talking about going from RNA to protein. So if you look at the word transcription, it has the word "script" in it, so I think of it as going from one written form to another kind of written form, and both use nucleic acid, so they both use this sort of alphabet, if you will, of nucleic acids. And so you're just going from one kind of alphabet to the next kind of alphabet. Translation, on the other hand, which is also the same term that we use when translating one language to another, describes going from nucleic acid to amino acid, so it's like you're using one kind of language and going to another kind of language, because you're going from nucleic acid building blocks to amino acid building blocks. So hopefully that helps you keep these terms straight a little bit. So what did we learn about the central dogma? Just remember the simple statement that DNA makes RNA, which makes protein. |
14 | Biomolecules | Amino Acids And Proteins1 | Central dogma - revisited | Hey. So you may have seen another video in which I talk about the central dogma of molecular biology, and this being the work of Francis Crick and James Watson, who delineated the structure of DNA and determined the flow of information to be from DNA to RNA to protein. And while this is the traditionally held view of the flow of information in living organisms, there have also been a lot of new discoveries that extend or even contradict the central dogma. And so I'm going to expand on some of those ideas here. And so this is the central dogma revisited. Before I go on and touch on those new ideas though, I would like to highlight the legacy of another famous scientist whose work was so, so critical to elucidating the double helix structure of DNA. And that was the work of Rosalind Franklin, seen here. And her contributions often go overlooked, of which I am just as guilty. And Franklin's work on the Xray diffraction images of DNA and her interpretation of that data were key to confirming the helical structure of DNA. And so whenever there's a mention of Watson and Crick, Rosalind Franklin deserves a shout out as well. So now on to the conflicting versions of the central dogma of molecular biology. As I said before, the traditional held view of the central dogma was that the flow of information occurs from DNA to RNA to protein. And DNA goes to DNA via replication, from DNA to RNA via transcription, and from RNA to protein via translation. And basically proteins perform virtually all the tasks within living cells and make up the very structure of those cells. It would be premature though to think that information flows in these pathways only and that proteins are the final expression of the information encoded in our DNA. So now that we have a firm foundation what the central dogma was as it was conceived originally, we can explore some of the newer discoveries that have been made that expand on this concept. And the first idea is that of reverse transcription, in which information flows backward, if you will, from RNA to DNA. And reverse transcriptase is an enzyme that generates complementary DNA or cDNA from an RNA template. And reverse transcriptase is needed for the replication of retroviruses, including HIV, is probably the most well known one. And these retroviruses use the enzyme to reverse transcribe their RNA genomes back into DNA, which is then integrated into the host genome and replicated along with it. So this is the first idea it was discovered back in 1970 that violated the central dogma. It was very unpopular at first, saying that information can actually flow from RNA back to DNA. Now another example of an alternate pathway for information flow is demonstrated by a group of viruses called RNA viruses. Now, these viruses have their genetic material stored as RNA, as opposed to DNA. And they can have their genome directly used by host cell replication machinery as if it were messenger RNA and then translated directly into protein. Or they can have their RNA serve as a template for another RNA strand, that is then used for protein translation. Some wellknown examples of RNA viruses are the coronavirus, which was responsible for the SARS epidemic; the influenza virus responsible for the flu, that you have to get a shot for every year, and also paramyxovirus, which is the virus responsible for measles. Now, the next idea that represents a deviation from the central dogma as it was originally conceived is the discovery of what is called noncoding RNA or ncRNA. Noncoding RNA is a functional RNA molecule that skips this last step of being translated into a protein and can directly perform functions within the cell as an RNA molecule. Now, two examples of functional RNA molecules that you might be familiar with are transfer RNAs and also ribosomal RNA, both of which are used for the translation of messenger RNAs into proteins. Now a final consideration that I'll be bringing up that sort of expands on the central dogma is the field of epigenetics, which is the study of heritable changes in gene activity that are not caused by changes in DNA sequence. So unlike in simple genetics where changes in phenotype are based on changes in genotype, epigenetics describes the mechanism where the same DNA sequence can be modified, resulting in a different phenotype without changes to the underlying DNA sequence. Now, some examples of mechanisms that produce such changes are DNA methylation and also histone modification. And this can help explain why you can have the same DNA in each of the cells of your own body, but those cells don't necessarily look or behave the exact same way. For example, the DNA contained in the nucleus of, say, one of your muscle cells is the same DNA that's contained in one of your skin cells, the same DNA in the nucleus of those cells, but these two cells are different because the expression of that DNA is modified by these epigenetic mechanisms. And these epigenetic mechanisms allow the transcription of only certain genes within the genome, depending on the type of cell. |
15 | Biomolecules | Amino Acids And Proteins1 | Peptide bonds: Formation and cleavage | Let's talk about the peptide bond. Now, proteins are formed from the folding of polypeptide chains. And polypeptide chains are formed by linking amino acids together. And these links are called peptide bonds. So before we can work our way up to the fullyformed and functional protein, we have to start at the very beginning by forming a peptide bond between the first two amino acids. So let's review the structure of an amino acid really quickly. Here we have our backbone. We have our amino group, our carboxylic acid group. Here is our alpha carbon. And then, the r represents our side chain. Now, peptide bonds are formed by the nucleophilic additionelimination reaction between the carboxyl group of one amino acid and the amino group of another amino acid. So let me show you what that looks like here. Let's have another amino acid drawn right here. So the electron pair on the amino group of the second amino acid comes over to form a bond with the carbonyl carbon of the first amino acid. You give off a water molecule in the process, and then you get your newlyformed dipeptide. And here is our newlyformed peptide bond. Now, remember that a peptide bond is just an amide bond that is formed between two amino acids. And you should also make note of the fact that this bond is a rigid and planar bond that is stabilized by resonance delocalization of this nitrogen's electrons to this carbonyl oxygen. So we can draw that out here. Remember that there is a lone pair of electrons on this nitrogen that can move here. And then, these electrons will move to this oxygen atom, which also has its own two lone pairs of electrons. So it can also be represented like this. And we'll have the formation of a double bond here and then an extra lone pair on the oxygen atom. So as you can see, the peptide bond with this resonance delocalization of electrons has a lot of double bond character. And because of this doublebondlike character, the peptide bond is a very rigid and planar one. But don't confuse this with thinking that an entire polypeptide chain would be a rigidlike structure because even though there isn't much rotation about the peptide bond you do still have for free rotation about these alpha carbon atoms here. So now, here we can see we have a dipeptide. And if we kept adding amino acids along in a chain here, we would have a polypeptide. Now, if we take a closer look at the backbone of this chain, we can see that there is a pattern formed by the atoms that form this backbone. And here, you have a nitrogen atom, the alpha carbon, and a carbonyl carbon. And then, it repeats with the nitrogen atom, the alpha carbon, and a carbonyl carbon. And you get a pattern that looks like this. And each time you add a new amino acid, the pattern just repeats. So that, whatever length of your polypeptide chain, you always start out with a nitrogen atom and you always end with the carbonyl carbon. And so this end of the backbone of the polypeptide chain is called the amino or N terminal. And then, this end of a polypeptide chain is called the C terminal. And then once, within a polypeptide chain, each amino acid is called a residue. So that's the formation of a peptide bond and a polypeptide chain. So now how do we go about breaking this peptide bond to get two amino acids again? Let's give ourselves just a little bit more room here to work, and we'll redraw a bond between two amino acids as a peptide bond here. And remember that here is our peptide bond just to highlight it for you. And we can break this peptide bond in a process called hydrolysis. So if we have hydrolysis of this peptide bond, then we go back to forming two free amino acids. The hydrolysis of a peptide bond is helped along by two common means, and those two means are with the help of strong acids or with proteolytic enzymes. So when we use strong acids, we call this acid hydrolysis. And acid hydrolysis, when combined with heat, is a nonspecific way of cleaving peptide bonds. So say you have a long polypeptide chain. And then, you throw this polypeptide into a pot with some strong acid, and then turn up the stove to add a little heat. Then, you would just end up with a jumbled up mix of amino acids as each of the peptide bonds gets cleaved. So the other way of cleaving a peptide bond is with proteolysis. And proteolysis is a specific cleavage of the peptide bond with the help of a special protein, an enzyme called a protease. So unlike acid hydrolysis, proteolytic cleavage is a specific process. And you can choose which peptide bonds you cleave because proteases are pretty picky about where they will cut, and many of them will only cleave peptide bonds between certain specific amino acids. One example of this is with the protease trypsin. Trypsin only cleaves on the carboxyl side of basic amino acids, like arginine and lysine. And interestingly, this is the same enzyme that is produced by our pancreas to help us digest food. So now say we have the following polypeptide chain and it can be any old, arbitrary polypeptide chain and say we add trypsin to the environment that this polypeptide chain is in. And here I'm just representing the amino acids as their abbreviated form. Now with the addition of trypsin, where would this polypeptide chain be cleaved? Well, remember that trypsin cleaves on the C terminus of arginine and lysine. Here we have an arginine, and this would be considered the C terminal of arginine, since it's closest to the C terminal of the polypeptide chain. So we would get cleavage here. And then, likewise, we would have cleavage on the C terminal of this lysine residue here. And so with this particular polypeptide chain, you would end up with three different fragments after the addition of trypsin since it cleaves in these very specific places. And there are many other examples of specific proteases that cleave in at certain parts of polypeptide chains. And you probably don't really need to memorize which proteases cleave after which amino acids, but you should probably remember that they are just specific means of breaking a peptide bond unlike acid hydrolysis over here, which is a very nonspecific way of cleaving a peptide bond. |
16 | Biomolecules | Amino Acids And Proteins1 | Special cases: Histidine, proline, glycine, cysteine | Hey. So welcome to the Amino Acids Show. And this show is going to be featuring just 4 of the 20 amino acids. And those amino acids are histidine, proline, glycine, and cysteine. And these four amino acids deserve sort of an extra time in the spotlight because they each have a side chain that sort of sets it apart from the rest. And so let's go through onebyone and see what exactly these side chains are all about. So first up we have histidine, and I've drawn the structure of histidine for you here. And here is the backbone of the amino acid. So this is the same for all the amino acids. And then, you see here is the side chain of histidine. So what is so special about histidine, then, with this side chain? Well, as it turns out, this side chain has a pKa of around 6.5. And this turns out to be really close to physiological pH, which is right around 7.4. So what does this really mean to have a pKa that's close to physiological pH? Well, recall that, at a pH below an amino acid's pKa, the amino acid will exist in a protonated or positively charged form. And at a pH above an amino acid's pKa, it will exist in deprotonated form. Now, since the physiological pH which is the pH of the fluid within our own bodies is roughly equal to the pKa of histidine, then histidine's going to exist in both protonated and deprotonated forms. So this makes it a particularly useful amino acid to have at the active site of a protein where it can both stabilize or destabilize a substrate. So next step we have proline and glycine. If we go ahead and take a closer look at proline, we have the backbone structure here just like all the other amino acids. But then, you can see that the side chain is this alkyl group that wraps around and forms a second covalent bond with the nitrogen atom of the backbone. And so we say that proline has a secondary alpha amino group. And so this is just referring to the fact that the side chain forms a second bond with the alpha nitrogen the nitrogen in the backbone of this amino acid. Now, let's come over here and take a look at glycine. Here we have the backbone of the glycine molecule. And then, here we have the side chain. And the side chain for glycine is the simplest of all side chains. It is just 1 hydrogen atom. And I've drawn it out in wedgeanddash form here to help emphasize how because the side chain of glycine is a hydrogen atom you have a duplication of atoms coming off of this carbon here the alpha carbon. And so now this carbon is no longer a chiral carbon. So we'll write that here. No chiral alpha carbon. And this kind of sets it apart from the rest of the amino acids because the rest of the amino acids do have a chiral carbon meaning optical activity under planepolarized light. And glycine is also considered to be very flexible because it just has this little hydrogen atom as its side chain. And so there's a lot of free rotation around this alpha carbon. So we also consider it to be very flexible. So why are these two amino acids groups together? Well, they both play a role in disrupting a particular pattern found in secondary protein structure called the alpha helix. And an alpha helix is just a coiled up polypeptide chain that kind of looks like this. Now, because of its secondary alpha amino group, proline introduces kinks into this alpha helix. And it ends up looking like this. And also, since glycine is so flexible around its alpha carbon, it tends to do the same thing. And thus both of these amino acids are known as alpha helix breakers. So last but not least, we have cysteine. And here's the backbone again. And then, here is our side chain. And the side chain for cysteine has a special thiol group. And all thiol is really referring to is the sulfur and the hydrogen at the end there. So cysteines have this neat little trick where, if they're in close proximity with each other within a polypeptide chain or even between two different polypeptide chains, then their side chains can form a bond together between the two sulphur atoms called a disulfide bridge. So let's bring up 2 cysteine amino acids here. And I've shown them as isolated amino acids, but remember that they are part of a greater polypeptide chain. And the formation of the disulfide bridge occurs separate from the backbone. It is just between the side chains. The cysteine at the top is flipped over to bring its side chain in close proximity with the second cysteine below it. And then, the bridge forms between the two sulphur atoms. So before we go over how a disulfide bridge is formed, let's do a quick little review of redox reactions. And really, what you want to remember is the mnemonic OIL RIG. And that's to mind you that, in oxidation, you have a loss of electrons. So oxidation is loss. And in reduction, you have a gain of electrons. So reduction is gain. So remembering that will help you understand the disulfide bridge formation. So going back to our 2 cysteines. If you look closely at their side chains, the thiols are existing in reduced form. So you're going to find these tholes in a reducing environment. Now, say those cysteines end up in an oxidizing environment. In that case, you would see the loss of these hydrogens and then the formation of a bond between these two sulphur groups, which looks like this. So this here is your disulfide bridge. So when do you see cysteines going solo, kind of like you see here in the separate thiol group form? And when do you see them forming these disulfide bridges? Well, it turns out that it depends a little bit on what the rest of the environment around them is like. And as it turns out, the exterior of the cell or the extracellular space is an oxidizing environment. So I'll write that down here. So the extracellular space will favor the formation of disulfide bridges. But in the intracellular space, you're more likely to find a reducing environment. So I'll write that down here. And the way that I like to keep this straight is that I kind of think of how the interior of the cell has these little molecules called antioxidants. And these antioxidants, which you can kind of tell by the name of it stifle any oxidizing reactions. And so they keep the intracellular space a reducing environment. So you might have seen cysteine spelled without an e, like this. And you're probably thinking to yourself, is it cysteine with an e? Is it cysteine without an e? Is it cystine? Which one is it? I'm so confused. There are actually two official ways of spelling cysteine. The version with the e refers to cysteine when it's in its reduced form. And the version without the e refers to cystine when it has been oxidized. And the way I remember this is by picturing that the e stands for electrons. And so you have the electrons when you're in the reduced form. And then, you don't have the e for electrons when you're in the oxidized form. So hopefully that helps you keep things straight a little bit. |
17 | Biomolecules | Amino Acids And Proteins1 | Amino acid structure | Hey, so we're going to be talking about amino acids, and specifically with regards to amino acid structure. And before we dive on into this topic, I think it's nice to kind of take a step back and take a bigpicture view of amino acids and kind of figure out where do they exactly fit in the grand scheme of biochemistry and specifically human metabolism? And I think the best way to do this is to take a realworld example of a human protein called hemoglobin. Now, what is hemoglobin? Well, hemoglobin is found within the red blood cells that flow all through our bloodstream. So here's my little red blood cell. And each red blood cell is chockfull of this hemoglobin protein. I'm just going to write it out as each Hgb here. And this hemoglobin protein is what is actually responsible for picking up oxygen. When this little red blood cell flows through the vessels and the lungs, picks up oxygen, and then transports this oxygen to all the various tissues within our bodies. And so you can kind of think of hemoglobin as a car of sorts. My favorite car is a Porsche 911. If you want to be more environmentally friendly, you could think of a Prius, or something like that. So whatever your car is, oxygen is like the passenger for that car. And so hemoglobin goes by the lungs, picks up oxygen, delivers it to the tissues. And then tissues are just groups of cells that are of a similar type, and so each of the cells in these tissues then takes the oxygen and uses it to generate adenosine triphosphate, or ATP, which is the energy source for all the various metabolic processes that go on within our cells to help keep us alive. So now where do amino acids fit into all of this? Well, amino acids are the building blocks of this hemoglobin protein. And so without amino acids, this entire vitally important process wouldn't be able to occur. Now, sticking with the car analogy just a little bit longer, just like we have different types of components that come together to form different types of cars, whether it be a Porsche or a Prius, what have you, you can have different types of amino acids. And there are 20 of them to be exact that can come together to form countless, countless different types of proteins. And so now that you have an idea of where amino acids fit in this bigger picture of a metabolic process, let's go ahead and take a closer look at what the actual structure of an amino acid is. First, we have the amino group. And then we have the carboxylic acid group. And already you can start to see where the name amino acid comes from. You have amino, from the amino group, and then you have acid from this carboxylic acid group here. And then linking the two groups is this carbon atom, which we call the alpha carbon. And then bound to the alpha carbon is a hydrogen atom as well as a unique side chain, or R group. We just use R to denote any generic side chain. So each of the amino acids has this same generic structure, and what makes each of the 20 amino acids different from each other is this R group, or the side chain. So each of the side chains for the amino acids is going to look different. One thing that's important to note is that this carbon atom, the alpha carbon, is also known as a chiral carbon. And what does a chiral carbon mean again? Well, a chiral carbon is a carbon atom that has four unique groups bound to it. So if we take a look at this carbon, we can see that one group that's bound is the amino group. Another group is the carboxylic acid group. The hydrogen atom makes the third group, and then the fourth group bound to it is the R group or the side chain. And so the alpha carbon in amino acids is considered a chiral carbon. And remember that chirality really refers to optical activity. In other words, if you were to shoot plain, polarized light at an amino acid, then because this carbon is chiral, it would rotate to that light. And so that's what chirality is really referring to. It's referring to optical activity. Now, it's important to note that there is one exception among the amino acids for chirality, and that is the amino acid glycine. And that's because the side chain, or R group, for glycine is just a hydrogen atom. It is the simplest of all side chains, just one hydrogen atom. And so if you were to substitute a hydrogen atom in place of this R group, you would see that you have a duplication of atoms coming off of this alpha carbon in the case for glycine. And so glycine is the only amino acid that does not have a chiral carbon. So that's just important to make note of. So let's give ourselves a little bit more room here. Now, another way that you can portray the structure of an amino acid is with something called a Fischer projection. And Fisher projections help to highlight the relationship of the four groups around a chiral carbon. So let me draw that for you here. First, you have your amino group, and up top you have your carboxylic acid. Here is your hydrogen atom, and at the bottom is the side chain, or R group. And just to orient you a bit, here in the center is the chiral carbon, the alpha carbon here. And then you have the four groups coming off of the chiral carbon. And the horizontal bonds here you can kind of picture those as coming out of the plane of the computer towards you. And then these vertical bonds here are coming out of the plane of the computer away from you. And this particular configuration is called an Lamino acid. And conversely, you can have the mirror image of this, which I'll draw for you here. And this particular configuration is called a Damino acid. And these two configurations are called enantiomers. And enantiomers are mirrorimage molecules that are not superimposable. So you can picture these are mirror images of each other, but if you were to take this Damino acid and try to superimpose it on the L, you wouldn't be able to do that. You can kind of think of these two configurations like your left and right hand, and although your left and right hand are mirror images of each other, you can't superimpose them on one another. And that's the relationship between an L and a Dconfiguration for Fischer projections. And these two configurations look awfully similar and are really easy to mix up, and so the way that I like to keep them straight is if I look at where the amino group is let's take the L amino acid. If I look at where the amino group is, I can see that it is to the left of the projection, so L is for left amino group. Now, if I look at the D amino acid, I again look for the amino group, and I see that it's to the right of this configuration. And so D, which actually means dextro, or right, in Latin, is for right amino group. That's kind of how I like to keep them straight. So why is it important to distinguish between L and Damino acids? Well, the L form of an amino acid is the only form that you will find within the human body, and so that's really important to remember that the Lconfiguration is the kind that you find within humans. All right. Now how about we review a little bit about everything that we learned? First, we sort of got a big picture of where amino acids fit in a larger metabolic process, such as in the example of hemoglobin. And then we learned about the structure of an amino acid and the fact that the central alpha carbon is a chiral carbon with optical activity, and the one exception to this rule is the amino acid glycine, which just has the simplest side chain of a hydrogen atom, and therefore it is not a chiral molecule. And then we also learned about the Fischer projections for amino acids and the fact that the Lconfiguration of an amino acid is the only one that you find within the human body. And there you have it. |
18 | Biomolecules | Amino Acids And Proteins1 | Isoelectric point and zwitterions | Hey, so we're going to be talking about the isoelectric point, or pI as it's abbreviated. Now, the isoelectric point is the point along the pH scale at which a molecule, and in this case we're going to be talking about an amino acid, exists in a neutral form with zero charge. In other words, it is neither positively or negatively charged overall. It is isoelectric, and "iso" means equal. And it's nice to know the isoelectric point for an amino acid, because then we can predict whether or not it will be charged at a certain pH. And who doesn't want the power of prediction? So how do we figure out the isoelectric point for an amino acid? Well, let's start with the generic amino acid structure here. So now let's take a look at the two functional groups on this amino acid. Ignoring the R group, or the side chain, for the time being, we're going to be talking about the amino group and the carboxylic acid group. So the amino group here, it has this nitrogen, which is a very happy proton acceptor. So we're going to write that here. And because it's a happy proton acceptor, it is considered to be basic. And we've drawn it out in its protonated form here after it's accepted an extra hydrogen, or proton. So now coming over to our carboxylic acid group, this group is a very willing proton donor. And because it is a proton donor, we call this acidic. And so we've drawn it out here after it's already donated its protons, so it has a negative charge. And now looking at the overall net charge of our amino acid, we can see that we have a positive charge here and a negative charge here, and so the overall charge is 0. And we have a special name for when you have a molecule that has both a positive and a negative charge present. And that special word is called a "zwitterion," which comes from the German word for "hybrid." So now what would happen if we take our amino acid and we put into a solution that is a very low pH, say a pH of 1? In other words, an acidic solution. Well, we can think of acidic solutions as having a lot of excess protons around. So anything that can be protonated on our amino acid is going to be protonated, and so it's going to look like this. And now if you take a look at both of the groups on our amino acid, you can see that our amino group is still in its protonated form and carries a positive charge. But now our carboxylic acid group has gained a proton and lost its negative charge. And now you can see that the overall net charge on this molecule is now positive 1. So now let's come over to the other end of the spectrum. Let's put our amino acid in a solution with a very high pH, say a pH of 12. And so this is going to be really basic solution, and we can think of really basic solutions as having a lot of excess hydroxide anions around. And so now, everything that can be deprotonated on our amino acid will be, so it's going to look like this. And if we look at our overall net charge of our amino acid now, our amino group has been deprotonated so now it is neutral, and the carboxylic acid group has been deprotonated and so it has a negative charge again. And so it has an overall net charge of negative 1. So now we know that we have a range of forms that our amino acid can take. We have the positively charged version at low pHs all the way up to the negatively charged version at high pHs. Now back to our question about the isoelectric point. So the isoelectric point is the pH at which we go from the positive to the negative form. In other words, it's where we find the zwitterion. And to find out the exact pH, we have to take the average of the pKa's of our two functional groups. And recall that the pK is just the negative log of the acid dissociation constant. So on average, and it varies between all the different amino acids, but on average, the amino group has a pK of around 9. And then on average, the pK for the carboxylic acid group is right around two. So now if we just give ourselves a little bit more room here, we can calculate what the pI, or isoelectric point, would be for our generic amino acid. So taking the average pK for the amino group and then the average pK for the carboxylic acid group, then we divide by 2, then you get 11 over 2. And we come to an isoelectric point of 5.5. But say our amino acid has a side chain or an R group that is also a functional group? Then, we would also have to take the pK for that group into account when we calculate the isoelectric point. So what have we learned? Well, we've learned that the isoelectric point is the pH at which a molecule's found in neutral form, in this case, when an amino acid is in its zwitterion form. And we also learned how to calculate this isoelectric point for an amino acid by taking the average of the pKs of all the functional groups in that amino acid. |
19 | Biomolecules | Amino Acids And Proteins1 | Classification of amino acids | All right. So let's go through the classification of amino acids. And I've highlighted the word class within classification for you, because I'm going to paint for you a picture of a classroom that is full of 20 different amino acids. And just picture this as the most diverse classroom you've ever seen, because each amino acid has their own unique side chain, and this makes them distinctly different from the amino acid next to them. And just like a real classroom full of kids, even though each amino acid is unique and special in their own way, you can start to see that some of these amino acids are more alike than they are different. And we can start to see these similarities in the chemical properties of the side chains, and this allows us to group them together into various categories. And those chemical properties include the charge of the side chain, the ability of the side chain to undergo hydrogen bonding, and also whether or not we can classify that side chain as being either acidic or basic. So the 20 amino acids can be split broadly into kind of two main groups. The first group includes the nonpolar amino acids, and then the second group includes the polar ones. And the nonpolar amino acids can also be thought of as the hydrophobic, or waterfearing, amino acids. And conversely, you have the polar ones. Those can be considered hydrophilic, meaning waterloving. And yet another way that I like to kind of think about these two main groups are the hydrophobic amino acids they're kind of like the waterhaters. They don't really want to interact with water at all. They'd rather just interact with themselves. Whereas the hydrophilic amino acids are very open and welcoming to interacting with water, and so they're waterlovers. And then within the two groups of nonpolar hydrophobic and polar hydrophilic amino acids, you then have a further breakdown into subgroups. And those subgroups include those amino acids that have alkyl side chains, aromatic side chains, neutral ones, acidic ones, or basic ones. So let's take a closer look at those amino acids that have alkyl groups as side chains. And as you can see here, we have seven different amino acids, and I've just drawn out the side chain for you. I've left the rest of the molecule out just to fit everything in here. And we have glycine, alanine, valine, methionine, leucine, isoleucine, and proline. And proline is the exception. I've drawn out the entire amino acid there, because as you can see, its side chain forms this interesting ringed structure with the amino group in the backbone of the molecule. So I just included it there for completeness. So all these side chains are made up of alkyl groups, with the one exception being glycine, because its side chain has only a hydrogen atom in it. But because it behaves similarly to an alkyl chain side group, it gets slumped into this category of amino acids. And whenever you see an amino acid with an alkyl group as its side group, you should be thinking that this amino acid is nonpolar. And so they're also going to be hydrophobic. Now, let's take a closer look at those amino acids that have aromatic groups as part of their side chain, and remember, we're still under the umbrella of nonpolar hydrophobic amino acids here. And so I've drawn out for you here two amino acids, phenylalanine and tryptophan. And what should you be thinking when you're looking at these amino acids? So besides thinking, oh, those amino acids must smell really good, because they're called aromatic amino acids well, that might be true, but you should also be thinking the same thing that you think when you see amino acids with alkyl groups as their side chains. These amino acids that you see here are also nonpolar and hydrophobic. And that kind of makes sense, because aromatic chains are also just made up of carbons and hydrogens. And you weren't wrong if you thought that aromatic compounds might smell really good, because many of our most aromatic herbs and spices that we're all familiar with, like basil or cinnamon and vanilla, are composed of the same sorts of ring structures that we see here. All right. So now that we've tackled the nonpolar hydrophobic amino acids, let's dive on into the polar and hydrophilic amino acids. The first group that we will look at is the neutral group. Here we have serine, threonine, asparagine, glutamine, cystine, and tyrosine. The way that I remember that these are the polar amino acids is that these amino acids have a side chain that contain an oxygen or a sulfur atom, which tends to hog all the electrons around them to create a localized negative charge over that atom and then a positive charge over the rest of the side chain. So you can kind of see why these amino acids like to hang out with water now, since water is also polar in the same way. And these amino acids are considered neutral, because although they are polar enough to interact with water, they're not strongly polar enough to qualify as an acid or a base. So which of the polar hydrophilic amino acids do qualify as acidic? Well, that would be these two amino acids here, aspartic acid and glutamic acid. As you can see, these amino acids have a carboxylic acid as part of their side chain, which is a very willing, strong hydrogen donor which qualifies these amino acids as acidic. When these side chains do donate their hydrogen and they're left in anion form, then in that case, we refer to them as aspartate and glutamate, respectively. So you might see them referred to in that way. Last but not least, we have the basic amino acids, and they're histidine, lysine, and arginine. And the way I remember that these amino acids are basic is that if you take a closer look at their side chains, you see a few nitrogen atoms. And remember that nitrogen is a very willing proton accepter, and this is why they qualify as basic. |
20 | Biomolecules | Amino Acids And Proteins1 | Four levels of protein structure | So why is it so important to learn about protein structure? Well, let's take the example of Alzheimer's disease, which affects the brain. So in certain people as they age, proteins and their neurons start to become misfolded and then form aggregates outside of the neurons, and this is called amyloid. So amyloid is really just clumps of misfolded proteins that look a bit like this. And as you can see, as this amyloid builds up, it starts to interfere with the neuron's ability to send messages, and this leads to dementia and memory loss. So if we can understand how these proteins become misfolded in the first place, then we might be able to find a cure for this debilitating disease. And to understand how proteins become misfolded, we must first understand how they become properly folded. So before we begin, I just want to do a quick review of terms. You can have one amino acid, so I'll just write AA for amino acid. And then you can have two amino acids that are linked together by a peptide bond. So this is a peptide bond. And as you add more and more amino acids to this chain of amino acids, you start to get what is called a polypeptide, or many peptide, bonds. And each amino acid within this polypeptide is then termed a residue. And then proteins consist of one or more polypeptides. And so I will use the terms polypeptide and protein interchangeably. So at the most basic level, you have primary structure. And primary structure just describes the linear sequence of amino acids, and it is determined by the peptide bond linking each amino acid. So if I were to take my amyloid example from Alzheimer's disease and I stretch out that protein all the way, then this linear sequence is just the primary structure. So then, moving on, we have secondary structure. And secondary structure just refers to the way that the linear sequence of amino acids folds upon itself. This is determined by backbone interactions. And this is determined primarily by hydrogen bonds. There are two motifs or patterns that you should be familiar with, the first of which is called an alpha helix. And if you were to take this polypeptide and wrap it around itself into a coillike structure, just like so, then you'd have the alpha helix. And the hydrogen bonds just run up and down, stabilizing this coiled structure. And another motif or pattern that you can be familiar with is with a beta sheet, and that just looks like this. It kind of looks more like a zigzag pattern. And the beta sheet is stabilized by hydrogen bonds, just like so. And if you have the amino ends and the carboxyl ends line up, like so, then this sheet is called a parallel beta sheet. And then conversely, if you have a single polypeptide that is then wrapping up upon itself just like this, and you have the hydrogen bond stabilizing like so, then you have the amino end coming around and lining up with the carboxyl end, and you have an antiparallel configuration. There is a third level of protein structure called tertiary structure, and tertiary structure just refers to a higher order of folding within a polypeptide chain. And so you can kind of think of it as the many different folds within a polypeptide, which then fold upon each other again. And so this depends on distant group interaction, so distant interactions. And just like secondary structure, it is stabilized by hydrogen bonds, but you also have some other interactions that come into play, such as van der Waals interactions. You also have hydrophobic packing, and also disulfide bridge formation. So if we explore hydrophobic packing just a little bit more over here say we have a folded up polypeptide or protein. And this protein is found within the watery polar environment of the interior of a cell. So if we have water on the exterior of this protein, then we will find all of the polar groups on the exterior interacting with this water. And then on the interior, you would find the nonpolar or hydrophobic groups hiding from the water. Disulfide bridges, on the other hand, describe an interaction that happens only between cystines. So cystines are a type of amino acid that have a special thiol group as part of its sidechain. And this thiol group has a sulfur atom that can become oxidized, and when this oxidation occurs, you get the formation of a covalent bond between the sulfur groups. The formation of a disulfide bridge happens on the exterior of a cell, and you tend to see the formation of separated thiol groups on the interior of a cell. And that is because the interior of the cell has antioxidants, which generate a reducing environment. And since the exterior of a cell lacks these antioxidants, you get an oxidizing environment. So if I were to ask you which environment favors the formation of disulfide bridges, you would say the extracellular space does. Then there is one final level of protein structure, and that is called quaternary structure. And quaternary structure describes the bonding between multiple polypeptides. The same interactions that determine tertiary structure play a role in quaternary structure. And so let's say I have one folded up polypeptide, two folded up polypeptides, and a third and a fourth. The quaternary structure is described by the interactions between these four polypeptides. And within the completed protein structure, each individual polypeptide is termed a subunit. Since this protein has four subunits, it is called a tetramer. And so if I were to have two subunits, it would be called a dimer, three would be called a trimer, and then anything above four is called a multimer. So the term for a completely properly folded up protein is called the proper conformation of a protein. And to achieve the proper confirmation, you must have the correct primary structure, secondary structure, tertiary structure, and quaternary structure. And if any of these levels of protein structure were to break down, then you start to have misfolding, which can then contribute to any of a number of disease states. |
21 | Biomolecules | Amino Acids And Proteins1 | Conformational stability: Protein folding and denaturation | Let's talk about conformational stability and how this relates to protein folding and denaturation. So first, let's review a couple of terms just to make sure we're all on the same page. And first we'll start out with the term conformation. And the term "conformation" just refers to a protein's folded 3D structure, or, in other words, the active form of a protein. And next, we can review what the term "denatured" means when you're talking about proteins. And denatured proteins just refer to proteins that have become unfolded or inactive. So all conformational stability is really talking about are the various forces that help to keep a protein folded in the right way. And these various forces are the four different levels of protein structure, and we can review those briefly right here. So recall that the primary structure of a protein just refers to that actual sequence of amino acids in that protein. And this is determined by a protein's peptide bonds. And then next, you have secondary structure, which just refers to the local substructures in a protein, and they are determined by backbone interactions held together by hydrogen bonds. Then you have tertiary structure, which just talks about the overall 3D structure of a single protein molecule. And this is described by distant interactions between groups within a single protein. And these interactions are stabilized by Van der Waals interactions, hydrophobic packing, and disulfide bonding in addition to the same hydrogen bonding that helps to determine secondary structure. And then quaternary structure just describes the different interactions between individual protein subunits. So you have the foldedup proteins that then come together to assemble the completed overall protein. And the interaction of these different protein subunits are stabilized by the same kinds of bonds that help to determine tertiary structure. So all of these levels of protein structure help to stabilize the foldedup, active conformation of a protein. So why is it so important to know about the different levels of protein structure and how they contribute to conformational stability? Well, like I said, a protein is only functional when they are in their proper conformation and their proper 3D form. And an improperly folded or degraded, denatured protein is inactive. So in addition to the four levels of protein structure that I just reviewed, there is also another force that helps to stabilize a protein's conformation, and that force is called the solvation shell. Now, the solvation shell is just a fancy way of describing the layer of solvent that is surrounding a protein. So say I have a protein who has all these exterior residues that are overall positively charged. And picture this protein in the watery environment of the interior one of our cells. Then the solvation shell is going to be the layer of water right next to this protein molecule. And remember that water is a polar molecule. So you have the electronegative oxygen atom with a predominantly negative charge leaving a positive charge over next to the hydrogen atoms. The same is true for each of these water molecules. So now as you can see, the electronegative oxygen atoms are stabilizing all of the positively charged amino acid residues on the exterior of this protein. So, as you can see, the conformational stability of a protein depends not only on all of these interactions that contribute to primary, secondary, tertiary, and quaternary structure, but also what sort of environment that protein is in. And all of these interactions are very crucial for keeping a protein folded properly so that it can do its job. Now, what happens when things go wrong? How does a protein become unfolded and thus inactive? Well, remember that this is called denaturation. And this can be done by changing a lot of different parameters within a protein's environment, including changing the temperature, the pH, adding chemical denaturants, or even adding enzymes. So let's start with what happens if you alter the temperature around a protein. And we can use the example of an egg when we put it into a pot of boiling water, because an egg, especially the white part, is full of protein. And this pot of boiling water is representing heat. And remember that heat is really just a form of energy. So when you heat an egg, the proteins gain energy and literally shake apart the bonds between the parts of the amino acid chains, and this causes the proteins to unfold. So increased temperature destroys the secondary, tertiary, and quaternary structure of a protein. But the primary structure is still preserved. So the takeaway point is that when you change the temperature of a protein by heating it up, you destroy all of the different levels of protein structure except for the primary structure. So now let's say you were to take an egg and then add vinegar, which is really just an acid. The acid in the vinegar will break all the ionic bonds that contribute to tertiary and quaternary structure. So the takeaway point when you change the pH surrounding a protein is that you have disruption of ionic bonds. And if we think about this a little bit more deeply, it makes sense, because ionic bonds are dependent upon the interaction of positive and negative charges. So when you add either acid or base, which in the case of an acid is just like adding a bunch of positive charges, you disrupt the balance between all of these interactions between the positive and negative charges within the protein. So now let's look at how chemicals denature proteins. Chemical denaturants often disrupt the hydrogen bonding within a protein. And remember that hydrogen bonds contribute to secondary, tertiary, all the way up to quaternary structure. So all of these levels of protein structure will be disrupted if you add a chemical denaturant. So let's take our same example of a protein with an egg, and say if you were 21 years older, you got your hands on some alcohol, and you added this to the egg, then all the hydrogen bonds would be broken up, leaving you with just linear polypeptide chains. And then finally, let's take our hard boiled egg from the temperature example and lets eat it. So here's my beautiful drawing of a person, representing you, eating this hardboiled egg. Once the egg enters our digestive tract, we have enzymes that break down the already denatured proteins in the egg even further. They take the linear polypeptide chain, whose primary structure is still intact, and they break the bonds between the individual amino acids, the peptide bonds, so that we can absorb these amino acids from our intestines into our bloodstream, and then we can use them as building blocks for our own protein synthesis. And that's how enzymes can alter a protein's primary structure and thus the protein's overall conformational stability. So what did we learn? Well, we learned that the conformational stability refers to all the forces that keep a protein properly folded in its active form. And this includes all of the different levels of protein structure as well as the solvation shell. And we also learned that a protein can be denatured into its inactive form by changing a variety of factors in its environment, including changing the temperature, the pH, adding chemicals or enzymes. |
22 | Biomolecules | Enzyme Structure And Function | Introduction to enzymes and catalysis | So today I want to talk to you about enzymes and how they're critically important pieces of cellular machinery. But first, let's review the idea that biochemical reactions happen in the body all the time. Almost every cellular process involves a biochemical reaction at one point or another. You know, the TCA cycle is actually just a series of different biochemical reactions in carbon metabolism. DNA replication, which needs to happen before a cell to go through mitosis, is also just a series of reactions. And this also applies to the expression of genes, going from DNA to RNA to protein. And we need enzymes because enzymes make all of these reactions go much faster. And let's look at this idea little more deeply, and how a reaction will go on differently when it has an enzyme versus not having one at all. So you may be familiar with the reaction where water and carbon dioxide can combine to form carbonic acid. And this is a reversible reaction, so it can go backwards and forwards. Now, when people make soda or any carbonated beverage, they'll start by pumping that soda can full of CO2. And while some of that CO2 will dissolve in the water and the can, the soda making companies are able to get a lot more CO2 in the water by using this reaction. The abundant CO2 will react with the water to form some carbonic acid in the can. And when you go to open the can, you'll hear a pop sound, which is really just a bunch of CO2 escaping. But after that, the soda will start to fizz really slowly. And what's happening here is the carbonic acid that was made before is slowly dissociating back to carbon dioxide in water as CO2 escapes. And that extra CO2 that's being made will come out of the soda solution, and you'll see it as little bubbles floating around. But what happens if you then take this person over here, and he'll pick up a can of soda and take a drink? That person might notice the soda will start fizzing a lot more once it hits his or her tongue. And this is because humans have an enzyme in their blood and saliva called carbonic anhydrase. And this makes the carbonic acid turn into carbon dioxide in water much more quickly. So more CO2 will come out of the can, and it will fizz more. And this is just one of the many examples of how enzymes make reactions go faster. So how exactly do the enzymes make the reactions go faster, though? Well, they use a bunch of different catalytic strategies to push reactions along a little more quickly. And I'm going to talk about a few those strategies just to give you an idea of what enzymes are doing. So first I'll mention acid/base catalysis, which happens when enzymes act like either acids or bases. Now, remember that acids and bases are proton donors and acceptors. And if you look at this type of reaction, which if you remember from organic chemistry is a ketoenol tautomerization reaction. We have a proton moving from a carbon atom to an oxygen atom. And since acids and bases are pretty good proton carriers, they could both help with this reaction, make it go a little more quickly, by helping to move that proton around, instead of this molecule of doing it by itself. Our next catalytic strategy is covalent catalysis, which happens when enzymes form a covalent bond with another molecule, usually their target molecule. Remember that covalent bonds involve two molecules sharing electrons. And looking at this reaction here, we have a decarboxylation reaction going on. Which, if you remember for organic chemistry, is when a carboxy or CO2 group is being taken off a molecule. And, if you remember, these reactions usually have a lot of electrons moving around. So if we had covalently bound enzyme that could hold on to some electrons, be an electron carrier, or what some people like to call an electron sink, then that would definitely help this type of reaction move a little more quickly. Next, we have electrostatic catalysis. Now, if you remember, DNA is a very negatively charged polymer because of all the negatively charged phosphate groups that we find in DNA. So if an enzyme had a metal cation on it, like magnesium, we could use it to stabilize the negative charge found in DNA and make it a little easier to work with. And DNA polymerase, which is the enzyme that allows DNA replication to occur, does exactly this. And in order for it to help with DNA replication, it needs to find a way to counteract all of the negative charge on DNA. The magnesium ions totally come in handy there. So the last catalytic strategy I want to mention is a little more general. And it has to do with proximity and orientation effects. Remember that in order for two molecules to react with each other, which is usually what enzymes help out with, they need to physically collide at some point. If we have molecule A and molecule B, they'll only react once they crash into each other. And a lot of enzymes are able to bring two molecules close together, so that these types of collisions happen more often, making the two molecules react more quickly. Also remember that the orientation of the two colliding molecules in space is also really important. If molecule A and molecule B collide, but one of them is upside down or not in the correct position, then the collision may not result in a successful reaction. So enzymes also make sure that the two molecules will collide in the right orientation. And all of this increases the frequency of collision in general, but also helps to make sure that those collisions are successful and result in a reaction. So what did we learn? Well, first we learned that the role enzymes play is to make biochemical reactions happen more quickly. And the next thing we talked about were four of the many different catalytic strategies that enzymes can use. We talked about acid base catalysis which helps with proton transfer. We talked about covalent catalysis, which helps with electron transfer. We mentioned electrostatic catalysis, which deals with stabilizing charge. And finally, proximity in orientation effects, which increase the frequency of successful collisions between molecules that we want reacting together. |
23 | Biomolecules | Enzyme Structure And Function | Enzymes and activation energy | Today, we're going to talk about how enzymes can influence a reaction's activation energy. But first, let's review the idea that enzymes make biochemical reactions go faster. And in order to do that, they use a bunch of different catalytic strategies. Now, there are lots of different catalytic strategies that enzymes use. But a couple of the key ones are acid/base catalysis, where enzymes use their acidic or basic properties to make reactions go faster by helping out with proton transfer. There's also covalent catalysis where enzymes covalently bind to a reacting molecule to help with the electron transfer. There's electrostatic catalysis where enzymes use charged molecules or metal ions to stabilize big positive or negative charges. And we also have proximity and orientation effects, where enzymes make collisions between reacting molecules happen a little more often. So what effect do these catalytic strategies actually have on a reaction? Well, let's look at a sample reaction where we're having molecule A being converted to molecule B. Now, we can look at the process of this reaction using something called a reaction coordinate diagram. And here, we'll plot the energy state of our molecules against the progress of the reaction. So essentially, using this graph, we'll follow the energy level of molecule A as it's converted to molecule B. Remember that a molecule's energy level is related to its stability. And something that has a lower energy state is more stable. And for something to transform to a more unstable form, it needs an input of energy to get there. So looking at this graph, you'll notice that the energy of molecule A will rise up pretty high and then drop all the way down to the energy of molecule B. And we can actually define a couple of values from this graph. The transition state of a reaction, which is represented by this double dagger symbol, is the highest energy point on the path from A to B. And it's where you'll find the most instability throughout the entire reaction. Now the difference between the energy level where we start and the top of our graph at our transition state is what we call the delta G double dagger or the free energy of activation. And this is the amount of energy that A needs to have in order to break the reaction barrier to ultimately get to point B. You'll also notice that there is a difference in energy between point A and point B. And we call this the standard free energy change for the entire reaction. And it represents the net change in energy levels between our reactant and our product. And it's also the energy that is released into the environment once the reaction is over. Reactions you typically look at will have their products at a lower energy state than their reactants since that makes the reaction spontaneous. Now, it's important to recognize that it is the free energy of activation energy value, which is the difference between point A and the transition state, that usually determines how quickly a reaction will go. And usually this energy value is much higher than the free energy change for the reaction, which is why enzymes speed up a reaction by lowering the reaction's activation energy. Now, I want to quickly point out that you may see delta G double dagger written out as EA in some textbooks. And you may see the standard free energy change for the reaction written out as E reaction. And I'm just letting you know that might see both sets of terms used from time to time. Now, let's look at an analogy to get a closer look at how this all works. And let's say there's a giant hill that you're trying to climb. And it's a pretty steep hill, that goes up really high. But you need to get to the other side of the hill. Now, this would be a pretty scary thing on its own since you would need to go all the way up and then all the way down the mountain to get to the finish line. But if I were to give you a shovel, then now you could dig your way through the mountain and not have to climb up so high. In this example, the shovel represents an enzyme and the hill represents the activation energy barrier that prevents you from getting to start to finish. By using the shovel, you're able to lower the height of the hill you have to climb. But in both cases, it's important to recognize that you still started and finished at the same points. So let's go back to our example from before with our reaction coordinate diagram. But now, let's say that the reaction has a catalyst. So with the catalyst, the activation energy barrier that molecule A has to overcome in order to get to point B is much smaller. And this will mean that your reaction will have a transition state with a much lower energy, meaning that it's more stable with the enzyme and also that the reaction as a whole have a much lower activation energy. Now it's really important to recognize that like our example where you're trying to climb the hill, the enzyme will not be changing the starting and ending points of the reaction. It doesn't change molecule A or molecule B. Your starting and ending points are always the same. And the only thing that changes is the path that you take to get from A to B. Now since our starting and ending points aren't changing, it follows that the enzymes are not used up when they catalyze a reaction. And there is no permanent change to the enzyme following a reaction. So what did we learn? Well, first we learned that enzymes work by lowering the free energy of activation of a reaction, making it much easier for the reactants to transition and form products. And we also learned that the free energy of the reaction doesn't really change when you use an enzyme and when you don't. Second, we learned that, despite the change in pathway to get from A to B, the reactants and products do not change when using an enzyme versus when not using an enzyme. And finally, we learned that enzymes are not consumed when they catalyze a reaction and the same enzyme can catalyze reactions over and over again. |
24 | Biomolecules | Enzyme Structure And Function | Induced fit model of enzyme catalysis | So today, I'm going to talk to you about the induced fit model of enzyme catalysis and how this concept can tell us a lot about how enzymes work. But before we do that, let's review the idea that enzymes make reactions go faster. And when you look at a reaction on a reaction coordinate diagram, you'd see that the catalyzed reaction would have a much smaller activation energy than the uncatalyzed one. Also remember that because of this, the energy of the catalyzed reaction's transition state is far lower than the energy of the uncatalyzed reaction's transition state. So what do enzymes look like? Well, most enzymes are proteins, or at least partially made up of protein. And substrates are any molecule that an enzyme will act on. And often, these substrates are the reactants that the enzyme will ultimately help turn into products through a reaction. Now enzymes also have what is called the active site, which is the location on the enzyme where substrates bind. And that's where the reaction ultimately happens. And it's important to recognize that every enzyme has a unique active site that will only bind to certain substrates. And just to clarify, I've referred to the active site here as both of the notches found on the enzyme, and not the space in between them. So here, of the two substrates I've drawn, the enzyme will only be able to bind to substrate 1, since they fit together like puzzle pieces. Whereas the shape of substrate 2 isn't going to fit nicely in the enzyme's active site. Now since enzymes have unique active sites, we say that enzymes are specific to certain substrates, and by extension certain reactions. But let's dive a little deeper into what happens when enzymes and substrates bind to each other and how that binding pattern changes as a reaction progresses. So first you'll have your enzyme here and your substrate over here. And I'm just going to label this with the number 1, since it'll be the first thing that happens in the sequence of events to come. And at this stage, nothing has happened yet. And the enzyme and the substrate have yet to come in contact. So next what will happen is the enzyme will bind to the substrate. But this binding won't be perfect. So we'll call this initial binding, which is stage 2 of the process. And what that means is that the forces holding these two together are strong, but they're not at their maximum strength just yet. And enzymes and substrates don't actually fit together quite like puzzle pieces. And they actually work a little bit more like two pieces of clay that will both mold together so that the fit is much tighter. So in our next step, this is exactly what happens. The enzyme and the substrate will both change shape a little bit and bind to each other really strongly. And we call this the induced fit because both the enzyme and the substrate have changed their shape a little bit so that they bind together really tightly. And it's at this point where the reaction that the enzyme is catalyzing is at full force. And this would be stage 3. So our next stage occurs after the reaction is completed and the binding becomes similar to what it was in stage 2. But the difference here is that there was something different about the substrate. So in this reaction, the enzyme is cutting our substrate into two parts. So now, the two parts have become separated. And this would occur after the reaction is finished. And we'll call this stage 4. Now in our next and last stage, the products of the reaction have been released from the enzyme. And our enzyme is back in the same state that it was in stage 1. And we'll call this stage 5. Now, let's look at this from a slightly different angle. I'm going to label the enzyme as E, the substrate as S, and our two products as P1 and P2. And they're going to represent this series of events, these different steps in the sequence of reactions. So first we'll have E and S separate. And this is stage 1. And next, E and S will bind to each other to form an enzyme substrate complex, which I have called ES. And it corresponds to stage 2 from before. Now what's really interesting is that in the next step, where we had the induced fit of stage 3, we're actually at the transition state of the entire reaction. And this is the same as that really high energy point that we saw at the beginning of this video. And it's at the point of the transition state where our enzyme is most tightly bound to its substrate. Now, I've written the substrate out here with the letter X. Because of the reaction's transition state our substrate isn't quite a reactant and it isn't quite our product either. It's somewhere in between. So that's why I've written it out as X instead of S. And I've also written this double dagger symbol, which is just a universal symbol for transition states. Now in our next stage, which is after the reaction has occurred, since it exists after the transition stage, we have the enzyme bound to the two products P1 and P2. And this was stage 4 from before. And then finally in our last stage, stage 5, we have our enzyme, which is now separated from our two products, P1 and P2. Now the big M away from this is that binding between enzyme and substrate is strongest at the reaction's transition state. And this is because the enzyme and the substrate have molded together. And that's why we call it the induced fit. Now, some enzymes will actually bind to more than one substrate. And if we look at a reaction that might be familiar, which is lactic acid fermentation, we can see that our enzyme, lactase dehydrogenase, will have space to bind to two different substrates in this reaction, one space being for NADH and the other being for pyruvate. So enzymes don't necessarily bind just to one substrate. Now, sometimes things will bind to enzymes at places other than their active sites. And we call this allosteric binding. So if we have an enzyme here with it's active site, a regulating molecule like an inhibitor made by the enzyme at a different location than the enzyme's active site. Now when something binds to an enzyme like this, it usually has the effect of changing the shape of an enzyme in some way to affect its ability to catalyze reactions. So in this case, when an inhibitor binds top the enzyme, it might change the shape of the active site, thereby inhibiting the enzyme, as it's no longer able to bind to its intended substrate. They don't quite fit together anymore. So while enzymes bind to reactive groups at their active sites, they can also bind to regulators at their allosteric sites. And allosteric sites just refer to any binding site outside of the active site. And remember allosterically binding molecules can either be activators or inhibitors, any regulating molecule. So what did we learn? Well, first we learned that enzymes are specific and that they can each bind to only specific substrates to catalyze specific reactions. Next, we learned about the induced fit model and how enzymes bind their substrates most tightly in the middle of a reaction at the reaction's transition state. And finally, we learned that enzymes have both active sites and allosteric sites, with active sites being where the reaction takes place and allosteric sites being where regulation takes place. |
25 | Biomolecules | Enzyme Structure And Function | Six types of enzymes | So today, we're going to talk about enzymes and all the different kinds of reactions that enzymes can catalyze. But before we do that, let's review the idea that enzymes make biochemical reactions go faster. And if you look at a reaction coordinate diagram, you'd notice that enzymes speed up reactions by lowering their activation energy. Now, enzymes are generally named for their reactions, which is convenient because it makes it a lot easier to remember what an enzyme does if someone gives you its name. And a great example of this is that one of the enzymes involved in DNA replication is called DNA polymerase, which is named as such because it acts on DNA and specifically makes polymers of DNA. Now, the suffix "ase" is usually just one that you find at the end of most enzyme names. Now, another great example is that the enzyme that catalyzes the first step of glycolysis, which you may remember is the reaction between glucose and ATP to form glucose6phosphate and ADP, is called hexokinase. And "hexo" refers to the number 6, which is a reference to glucose being a sixcarbon sugar. And "kinase" is a term referring to enzymes that add phosphate functional groups to different substrates. So overall, hexokinase adds phosphates to sixcarbon sugars like glucose. Now generally, every enzyme has a very specific name that gives insight into the specific reaction that that enzyme can catalyze. So we can actually divide most enzymes into six different categories based off the kinds of reactions that they catalyze. Now, our first group is the transferase group. And the basic reaction that transferases catalyze are ones where you move some functional group, X, from molecule B to molecule A. And a great example of one of these reactions occurs during protein translation, where amino acids bound to tRNA molecules are transferred over to the growing polypeptide chain. So in this case, A refers to our amino acid chain, B refers to our tRNA, and X refers to this lysine residue, which is being transferred from B to A. And this reaction in particular is catalyzed by an enzyme called peptidyl transferase, which is an appropriate name since it is a transferase involved in making peptides. Next we have the ligase group, which catalyzes reactions between two molecules, A and B, that are combining to form a complex between the two, or AB. And an example of a reaction using a ligase that you might be familiar with occurs during DNA replication, where two strands of DNA are being joined together. So in this reaction, A and B represent the two separated DNA polymers, which are being joined to form a single strand. And this reaction in particular is catalyzed by an enzyme called DNA ligase, which is named since it's a ligase that works on DNA strands. Now our third group as the oxidoreductase group, which is a little different from the others since it actually includes two different types of reactions. And these reactions involve transferring electrons from either molecule B to molecule A or from molecule A to molecule B. Now, we say that an oxidase is directly involved in oxidizing or taking electrons away from a molecule, while a reductase is involved in reducing or giving electrons to a molecule. And we call these enzymes oxidoreductases together because they can usually catalyze both the forward and reverse reactions, which is why I used equilibrium arrows here instead of just a normal singleheaded arrow. Now a great example of an oxidation reduction reaction occurs during lactic acid fermentation, where electrons are either passed from NADH to pyruvate or from lactic acid to NAD. Now, this reaction is catalyzed by an enzyme called lactate dehydrogenase. Remember that the word "dehydrogenase" refers to the removal of a hydride functional group. And that's the same as saying the removal of electrons, since hydrides are basically just hydrogen atoms with two electrons on them instead of just one. Now, this enzyme is given its name since it's able to remove a hydride, or remove electrons, from a molecule of lactic acid. Next, we have the isomerase group. And enzymes in this group are typically involved in reactions where a molecule, like molecule A, is being converted to one of its isomers. And an example of this type of a reaction is the conversion of glucose6phostate to fructose6phosphate, which is one of the steps of glycolysis that you may remember. Now, this reaction is catalyzed by an enzyme called phosphoglucose isomerase, which is appropriately named since it creates isomers of glucose molecules that are phosphorylated. Now, our next category is the hydrolase category. And hydrolases use water to cleave a molecule, like molecule A, into two other molecules, B and C. And a great example of one of these reactions is the hydrolysis reaction that can occur to peptide bonds. And if we have this lysinealanine dipeptide here, it could be reacted with water to form two individual amino acids that are no longer bound. And this particular hydrolysis reaction can be catalyzed by a class of enzymes that we call serine hydrolases, which some people call serine proteases. And they are named this way because they are hydrolases that use a serine residue as the key catalytic amino acid that is responsible for breaking the peptide bond. Now, our last category is a little more complicated than the others. And it's the lyase group. Now, lyases catalyze the dissociation of a molecule, like molecule A, into molecule B and C, without using water like hydrolases would, and without using oxidation or reduction like an oxidoreductase would. And one example of a reaction catalyzed by a lyase is the cleavage of argininosuccinate into arginine and succinate. And this reaction takes place during the urea cycle, which you also might be familiar with. Now this specific reaction is catalyzed by an enzyme called argininosuccinate lyase, which is appropriately named because it is a lyase that catalyzes the breakdown of an argininosuccinate molecule. Now, it's important to recognize that since lyases don't use water or oxidation to break a bond, they need to generate either a double bond between two atoms or a ring structure in a molecule in order to work. So what did we learn? Well, first we learned that enzymes are sometimes named for their reactions. And next we learned about the six different types of enzymes. We have transferases, which transfer functional groups from one molecule to another; ligases, which ligate or join two molecules together; oxidoreductases, which move electrons between molecules; isomerases, which convert a molecule from one isomer to another; hydrolases, which break bonds using water; and lyases, which break bonds without using water and without using oxidation. |
26 | Biomolecules | Enzyme Structure And Function | Co-factors, co-enzymes, and vitamins | Today, we're going to talk about cofactors and coenzymes and how sometimes they can be essential to proper enzymatic function. But first, let's review the idea that enzymes make reactions go faster. And they do this by lowering the activation energy peak of their respective reactions. Let's also review the idea that enzymes bind their substrates at a location on the enzyme called the active site, which is where most of the reaction takes place. Now, not all enzymes are able to catalyze reactions on their own. And some need a little help. So if we have our enzyme here, trying to react with our substrate over here, sometimes something called a cofactor or a coenzyme will be needed, which will also need to bind to the enzyme in order for it to function properly. And we're going to go over what coenzymes and cofactors are and exactly how they work. So first, we'll talk about what a coenzyme is. Well, coenzymes are organic carrier molecules. And what I mean by "organic" is that they're primarily carbonbased molecules. And by "carrier," I mean that coenzymes hold on to certain things for an enzyme to make the catalysis run a little more smoothly. And a great example of a coenzyme is NADH, which acts as an electron carrier. And here, I've shown NADH dissociating into its oxidized form, NAD+, as well as to a hydride ion, which basically just exists as a pair of electrons that some other molecule would be grabbing. So NAD+ can accept electrons, causing the molecule to be converted to NADH, which could then carry electrons for an enzyme. Now if you remember the lactic acid fermentation reaction, where pyruvate is converted to lactic acid, you'd see that the enzyme catalyzing this reaction, lactate dehydrogenase, uses NADH as a coenzyme in order to transfer electrons to the pyruvate molecule, in order to turn it into lactic acid. And in this sense, NADH is acting as an electroncarrying coenzyme. Another example of a coenzyme is coenzyme A, which like NADH acts as a carrier molecule. But instead of carrying electrons like NADH does, coenzyme A, which we sometimes call CoA, holds on to acyl or acetyl groups instead. And you'd see CoA appear quite often in metabolic reactions, where it will carry these two carbon acetyl groups from one molecule to another. Now, cofactors are a little different from coenzymes. While coenzymes are only really involved in transferring different things from one molecule to another, cofactors are directly involved in the enzyme's catalytic mechanism. They don't strictly carry something like a coenzyme would, but might be stabilizing the enzyme or the substrates or helping the reaction convert substrates from one form to another. A great example of this is with the enzyme DNA polymerase. Remember that DNA polymerase is responsible for helping out with synthesizing new DNA during DNA replication. Now, you may remember that DNA is a very negatively charged molecule because of all the negatively charged phosphate groups that you'll find around it. Well, DNA polymerase uses a magnesium ion as a cofactor, which can use its big positive charge to stabilize all that negative charge on DNA. And you can see how this is different from a coenzyme. Becomes instead of acting as a carrier molecule, the magnesium ion cofactor is stabilizing the DNA and is more directly involved in the actual catalysis. Now, interestingly, what people normally called vitamin and minerals, like the kinds that a doctor would tell you to make sure you get enough of in your diet, are often different cofactors and coenzymes. And what's special about vitamins and minerals is that your body can't build them up from scratch. And you need to get them from your diet in order to stay healthy. So when we say vitamins, we typically refer to organic cofactors and coenzymes. So two great examples are ones we just discussed. Vitamin B3, which you may see being called niacin on a food label, is actually just a precursor for NAD. And vitamin B5 is just a precursor for coenzyme A. Minerals, on the other hand, are inorganic, meaning they aren't carbon based. And minerals are usually just cofactors in our body. So magnesium would be a great example of a mineral cofactor that an enzyme like DNA polymerase would use. Now, not all minerals act only as cofactors. Some minerals, like calcium, which can act as a cofactor, is also a critically important component of bone and teeth. And it doesn't strictly act as an enzyme cofactor here. It's actually an important part of the structure itself. So what did we learn? Well, first we learned that not all enzymes are able to function alone and some need a little help. And next, we learned that this help can come from coenzymes, which usually act as carrier molecules, or cofactors, which directly assist with the catalysis that the enzyme is doing. And finally, we learned that the vitamins and minerals generally refer to dietary cofactors and coenzymes. |
27 | Biomolecules | Enzyme Structure And Function | Enzymes and their local environment | So today, we're going to talk about the effects of the environment on enzymes and how a changing environment can affect an enzyme's ability to catalyze a reaction. But first, let's review the idea that enzymes make reactions go faster. And looking at a reaction coordinate diagram, you'd notice that enzymes speed things up by lowering a reaction's activation energy. Now, it's important to recognize that enzymes work best in specific environments. And when I say environment, I can be really referring to many different aspects of an enzyme's surroundings. But right now, we're really only going to be focusing on pH and temperature values. So let's take another look at this by imagining that we have this person over here. And he's hungry, so he's eating some food. Now, there are a bunch of different digestive enzymes in this guy's body that are going to help him break down all the food he's eating into tiny usable parts. So first the food will be in his mouth. And one of the enzymes found inside a human's mouth is called alpha amylase, which is responsible for breaking down complex carbohydrates like starches into small simple carbohydrates like individual sugars. And alpha amylase is able to work well in your mouth since it functions best at a pH of around 7, which is about the same pH as a human's mouth. Now moving along, the food that our guy ate is going to go all the way down to his stomach, where a whole different group of enzymes will start breaking down the food. Now, one enzyme that humans have in their stomachs is called pepsin, which breaks down big proteins into smaller peptides. Now, pepsin will be most active at a pH of around 2, which is also the pH of your stomach, which is so low because of all the stomach acid that you'd find there. Now in terms of temperature, both of these enzymes typically work at a temperature of around 37 degrees, which is the same as body temperature. But you can see that these two different enzymes are functioning at different environmental conditions. So what would happen if we took an enzyme and moved it into a different environment? Well, let's first look at the effects of changing the pH of an enzyme's environment and jump right in with an example. So remember that DNA is a very negatively charged molecule because of all the negatively charged phosphate groups that you'd find on DNA. And in order for the enzyme DNA polymerase to help out with DNA replication, it binds a magnesium ion cofactor, which it uses to stabilize all the negative charge on DNA. Now under normal pH conditions, the DNA polymerase hold onto that magnesium ion through an electrostatic interaction between magnesium and one of its aspartate residues, which would be deprotonated and thus negatively charged at neutral pH values. Now, if we were to take DNA polymerase and put it into an environment with a reduced pH, then that aspartate residue will become protonated since the pH has dropped so much. And in its protonated form, aspartate no longer has a negative charge and can't hold on to that magnesium ion anymore. And overall, this means that DNA polymerase won't be able to do its job properly in a low pH environment. And keeping this enzyme at an appropriate pH is essential to its normal function. So now, let's take a look at the effects of temperature changes on enzyme function. So remember that proteins need to fold into their secondary, tertiary, and possibly quaternary structures in order to be in their functional form. And significant changes to a protein's temperature can disrupt a protein's folded geometry and cause it to lose its functionality. If we have our same person from before, who was really hungry and really wants to eat, but now this person get sick with a fever, his temperature will rise. And a bunch of the digestive enzymes in his body will get all jumbled up and won't be properly folded anymore. And this is why you might have a hard time eating and digesting food when you have a fever. And this can sometimes lead you to throwing up anything you'll eat since all of the digestive enzymes in your body won't work anymore because of the increase in your body's temperature. And the food you eat will just sit there, sit there in your body and make you feel sick. So what did we learn? Well, first we learned that enzymes generally function only under very specific environmental conditions. And different enzymes will often function ideally in different environments from other enzymes. And next, we learned that changes to an enzyme's environment, like changes to the surrounding pH or temperature, can lead to a loss of enzyme functionality. |
28 | Biomolecules | Enzyme Kinetics | An introduction to enzyme kinetics | So we're going to talk about enzyme kinetics today, but first let's review the idea that enzymes speed up reactions by lowering the delta G of the transition state, or lowering the activation energy of a reaction. And also remember that for this to happen the reacting substrate, which I called S, will bind to the enzyme E to form the enzyme substrate complex ES before being turned into product P. Also remember that enzymes aren't used up when they catalyze reactions, and that's why we have this E at the end of the equation. Now, there's a general method of thinking that we want to use when talking about enzyme kinetics, and that's what I want to talk about today. Now, when we think about kinetics, we want to simplify a reaction as a change from A to B. And you may remember that the rate of this change from A to B would be equal to sub rate constant K which is dependent on the environment of the reaction, multiplied by the concentration of our starting material A. So for our sequence that I mentioned before, E plus S going to ES going to E plus P we have two reactions going on, one and two, which would each have their own rate equation. Rate one would be equal to rate constant K1 times the two starting material concentrations, which are E and S, while rate two would be equal to K2 times the concentration of one starting material ES. Notice that the reaction one has two reactants, E and S, while reaction two only has one, which is the ES complex. So what do we mean when we say "rate"? Well, the rate of a reaction is the speed that the reaction goes at, and we could also call it V, which is the symbol for speed. And for our entire reaction of transitioning S to P, our product, the speed would be equal to the rate of change of our concentration of product with respect to time, or for those of you who aren't really big fans of calculus, the change in P, delta P, over a change in time, delta T. So to increase the rate that we get new product, we could do this by either increasing the substrate concentration or by increasing the enzyme concentration, since we're going to assume that the K value is constant and can't be changed. Now when we think about enzyme kinetics we like to assume that we're in a situation where the total concentration of enzyme is constant. And this is generally the case when we're looking at enzymes working in different cells. Now if we say that we only have four enzymes here, and each enzyme can work at a speed of about 10 reactions per second, that would mean that the absolute maximum rate or our reaction would be 40 reactions per second. And this rate we would call "Vmax" or "max speed". And the idea here is that at really high concentrations of substrate the enzymes will be saturated and full up with substrate, and won't be able to react any more quickly. And even if we were to really increase the concentrations of substrate a lot, there will still be a Vmax. There's only so much that we can increase the rate of a reaction by increasing the substrate concentration. If we were to look at a graph and plotted the reaction rate V versus our concentration of substrate, we would see that as our substrate concentration got really, really high, the rate would level off as it approached our Vmax value. So when we think about enzymes and their kinetics this way we have made a couple of assumptions about how our enzymes and substrates are behaving, and I want to talk about these for a moment. The first assumption we have made is that our solutions are behaving ideally, and that we can actually classify our enzymes reaction into two distinct snaps, the first being the binding of substrate to enzymes, and the second being the transitions from substrate to product with the enzymes help. And by assuming that our solutions are behaving ideally and that we don't have any external factors messing things up, we can simplify our discussion of kinetics quite a bit. Our second assumption is that our two big constants stay constant. We're assuming that our enzyme concentration isn't changing from things like protein synthesis and degradation, and we're also assuming that our rate constant K isn't changing from environmental factors like changes in temperature. Our final assumption is that for our reaction substrate isn't forming product without the help of enzyme at a big enough rate for us to consider, and that it's negligible. Remember that enzymes only speed up reactions, so while it's possible for the substrate to form product without enzyme, we're going to assume that it's not really happening when we're talking about enzyme kinetics. So what did we learn? Well first we learned that we can classify enzyme catalysis into two important steps. The first is that the enzymes bind the substrate, and then second the formation of product, and we talked about how each of these steps has a distinct rate. Second, we learned that if we keep the enzyme concentration constant, then there will be a maximum speed, Vmax, for that reaction. |
29 | Biomolecules | Enzyme Kinetics | Steady states and the Michaelis Menten equation | Today we're gonna talk about MichaelisMenten kinetics and the steadystate. First, let's review the idea that enzymes make reactions go faster and that we can divide the enzymes catalysis into two steps. First the binding of enzyme to substrate and second the formation of products. Each of these reactions has its own rate. Let's also review the idea that if we keep the concentration of enzyme constant then a really high substrate concentrations will hit the maximum speed for a reaction which we call Vmax. First we'll talk about the SteadyState Assumption and what that means. Like I said before there are two steps to an enzyme's catalysis. Now when we use the term steadystate what we mean is that we're at a point where the concentration of ES or enzyme substrate complex is constant which means that the formation of ES is equal to the loss or dissociation of ES. Now notice that I've used equilibrium arrows between these steps and that was to show the idea that these reactions like any reaction can go forwards or backwards. Our enzyme substrate complex doesn't have to form products. It could just as easily dissociate back to an enzyme and a substrate molecule. I'll call these reverse reactions minus one and minus two. If we look at that in terms of our rates we can say that the rate of formation of ES would be the sum of rate one and rate minus two since both of these reactions lead to ES and the rate of loss of ES is equal to the sum of rates minus one and two since both of these lead away from ES. Now I also remember that products very rarely go back to reactants since these reactions are usually thermodynamically stable. Rate minus two is going to be so small in comparison to rate one that we can really just cross it out. Which means that we can swap out that second double headed for a single headed arrow. Using this information let's do some math. Now I'm going to be deriving a new equation. This can get a bit confusing so don't worry if you have a little trouble with this. Just rewind the video and try watching it a couple more times if you need to. I'll start up by drawing the same sequence I did before with the three different reactions, and I'll also write out that steadystate equation I mentioned before where we have rates forming ES equal to rates taking away ES. Now first thing I'll do is swap out those rate values for their rate constants times the reactants for those reactions. Rate one will be equal to K one times E times S and so on for the other two. Next I'll introduce a new idea and say that the total amount of enzyme available which we'll call ET or E total is equal to the free enzyme E plus the enzyme bound to substrate or ES. Using this equation I'm going to rewrite the E on the left side of our equation as the total E minus the ES which would be equal to the E we had there before. On the right side of the equation I just factored out the common term ES. Next I'm just going to expand the left side of the equation so take a moment to look at that. Now what I'm going to do is I'm going to divide both sides of the equation by K one. K one will disappear on our left side and on our right side I've put K one in with all the other rate constants. Now since all these rate constant are constant values I'm going to combine them in this expression of K minus one plus K two over K one into a new term KM which I'm going to talk a little bit more about later. In this next line I've done two things. First I've thrown in that KM value that I just mentioned, but I've also added ES times S to both sides of the equations and thus moved it from the left side to the right. In the next line I've done two things. First I switched the left sides and right sides of the equation just to keep things clear, but I've also factored out the common term ES on our new left side. Then what I'm gonna do is I'm gonna divide both sides of the equation by KM plus S so I can move that term to the right side. I'll make some more room over here and now what I'm gonna do is remind you that the speed of our whole process which I'll call Vo is equal to the rate of formation of our product which we called rate two before which is also equal to K two times ES. Now using our equation over here I'm gonna multiply both sides of the equation by K two. Here's where it gets really tricky. Remember that if we're if at our max speed so our reaction speed Vo is equal to Vmax which happens when our substrate concentration is really high, then our total enzyme concentration is going to be equal to ES since all of our enzyme is saturated by substrate and there won't be any free enzyme left. K two times ET instead of times ES would be equal to Vmax instead of being equal to Vo like you see at the top. I'll make some room here and then sub in K two ES for Vo and K two E total for Vmax and then we finally get to our end equation which is called the MichaelisMenten Equation and is super important when we talk about enzyme kinetics. Let's take a few steps back and talk about the Michaelis constant. First I'll write out the MichaelisMenten equation and if you remember we created this new term which I called KM, but we never really talked about what it meant. Let's get to that. Now if you bear with me for a moment and pretend that KM is equal to our substrate concentration then we can sub in that value into our MichaelisMenten equation which would put two S on the bottom, the sum of S plus S and then the S will cancel out and will be left with Vmax over two. What this means that KM which we call the Michaelis constant is defined as the concentration of substrate at which our reaction speed is half of the Vmax. When Vo is equal to 1/2 of Vmax. If we look at that on a graph from before you'd see that KM is a substrate concentration specific to our circumstances. Where our rate is at half of its max and the lower our KM, the better our enzyme is at working when substrate concentrations are small. We can use this KM term to quantify an enzyme's ability to catalyze reactions which we call Catalytic Efficiency. I'll rewrite the MichaelisMenten Equation. Remember we defined KM as a substrate concentration where Vo is 1/2 Vmax. Since it's a concentration it will be in units of molar or moles per liter. Now I'm going to throw a new term at you called Kcat which is equal to the maximum speed of a reaction divided by the total enzyme available. We call this the enzyme's turnover number. All these term is, is how many substrates and enzyme can turn into product in one second at its maximum speed. We measure it in units of seconds minus one or per second as in reactions per second. We can define an enzyme's catalytic efficiency as a combination of KM and Kcat, and we do this by saying it's equal to Kcat over KM. A higher Kcat or a lower KM would result in an increase in an enzyme's catalytic efficiency. Every different enzyme has a different catalytic efficiency in certain conditions. We can use this term to score enzymes on how good they are. We covered a lot of content in this video but the really crucial points to remember are first the idea of the SteadyState Assumption that we make when looking at enzyme kinetics. This is where we assume that the ES concentration is constant. We knew that the formation and loss of ES are equal. Second, we derived the critically important MichaelisMenten Equation which you should consider committing to memory. Third, we talked about how you can score how good an enzyme is at speeding up reactions by looking at that enzyme's catalytic efficiency which is a combination of two new terms we learned about Kcat and KM. |
30 | Biomolecules | Enzyme Kinetics | Cooperativity | So, we're gonna talk about Cooperative Binding, which is a very interesting topic when discussing enzyme kinetics. But first let's review the idea that we can divide enzyme catalysis into two steps. First, the binding of substrate to enzyme, and second, the formation of product. In using this idea we can derive the Michaelis Menten equation, which is very useful for quantitatively looking at enzyme kinetics. Also remember that as you increase substrate concentration, the speed of product formation will level off at it's maximum value as shown on this graph. Now the first thing that I want to talk about is that some proteins can bind more than one substrate, and not all enzymes have just one active site. So, E plus S can form ES through what I've called reaction of one, but some enzymes can react with another molecule substrate to form ES two, through what I've called reaction two. And again, to form ES three, through reaction three, and so on. Now, these enzymes can form product at any stage of this process no matter how many molecules of substrate are bound. Now, you would expect the rate of reaction one to be faster than the rate of reaction two. If we're looking at the example of an enzyme with three substrate binding sites, there are three empty sites available for substrate to bind through reaction one, and only two available for reaction two. So, you would expect rate one to be faster. Similarly, rate two would be faster than rate three for the same reason. And the idea is that the active site saturation does not increase with substrate concentration linearly. Now I get that that can be a mouthful so let's look at it graphically. But, we're also gonna show an exception to this rule. So in this first graph, I've plotted substrate concentration against the percent saturation of the enzyme. And as you can see the curve levels off as substrate binding sites become occupied. It becomes difficult to bind more substrate molecules as you have more substrate molecules bound. Next, I'm going to draw a different curve that you also might see in some enzymes and, that's where substrate binding happens more quickly as binding sites become occupied. Substrate binding changes substrate affinity. And we call this Cooperativity. Now with respect to cooperativity, we can define three new ideas: Positively Cooperative Binding occurs when substrate binding increases the enzyme's affinity for subsequent substrate. Negatively Cooperative Binding occurs when substrate binding decreases the enzyme's affinity for subsequent substrate more than you would normally expect. And NonCooperative Binding is the same as the first example where substrate binding does not affect the enzymes affinity for substrate molecules. So, let's look at this graphically. If we have a protein with let's say five binding sites, and plot the Fraction Occupied versus the Substrate Concentration, you would come up with three possible curves. The green curve, which takes on a sigmoidal shape, would represent an enzyme with Positive Cooperativity. The blue curve, with a hyperbolic shape, would represent an enzyme with NonCooperative Binding. And the red curve would represent an enzyme with Negatively Cooperative Binding. Now remember that the effects of cooperative binding are only seen after some substrate has already bound. Which is why the difference in the fraction occupied between the three curves is much smaller, it's smaller values, like the onefifth that I've shown here, than it is at the higher values. So, let's look at a specific example of a couple of proteins. So haemoglobin, or Hb, is the oxygen carrying molecule that you find in human blood, and it can bind up to a total of four oxygen molecules, and it exhibits Positively Cooperative Binding. Myoglobin, on the other hand, which is the oxygen carrying molecule that you find in muscle tissue, can only bind one oxygen molecule in total. And since it can only bind one, it must exhibit NonCooperative Binding since there's no subsequent substrate to speak of. Now, if we make a graph where we plot the fraction of active sites bound in each of these proteins versus the pressure of oxygen, remember oxygen is our substrate here, and since it's a gas we're gonna use pressure instead of concentration, you can see that the red sigmoidal curve associated with haemoglobin's positive cooperative binding looks different from the blue hyperbolic curve associated with myoglobin's noncooperative binding. So, what did we learn? Well, first we learned that some proteins can bind more than one equivalent of substrate. And next, we learned that there are three different types of Cooperativity: Positive, Negative, and NonCooperative. Finally, we learned about proteins that exhibit two different types of cooperativity, which were the oxygen binding molecules haemoglobin and myoglobin. |
31 | Biomolecules | Enzyme Kinetics | Allosteric regulation and feedback loops | So, today we're going to talk about how allosteric regulation can affect enzyme kinetics. But first, let's review the idea that an enzyme's catalysis can be divided into two steps. First, the binding of enzymes to substrate, and second the formation of products. And using this information, we can derive the MichaelisMenten Equation, which allows us to look at an enzyme's rate of product formation with respect to substrate concentration. Also remember substrates will typically bind to enzymes at the active site. So what do we mean when we say allosteric regulation? Well, we know that enzymes usually have an active site where substrates combined, but enzymes can also have what we call an allosteric site. And these allosteric sites are places on the enzyme where any enzyme regulator can bind. And I've put this star here just to point out that allosteric sites can be anywhere on a enzyme. There can be any number of them as well. So what do we mean when we say regulators? Well, we generally say there are two types of regulators. There are allosteric activators, which increase enzymatic activity and activate them, and allosteric inhibitors, which decrease ezymatic activity and inhibit the enzymes. So let's take a look at what we mean by increasing and decreasing ezymatic activity from a kinetic perspective. So, remember the MichaelisMenten equation, and if we're assuming substrate concentration to be constant, then there are two ways to influence enzymatic activity, or VO. In this first graph, I've drawn three different curves. The blue curve represents the enzyme functioning without an allosteric regulator at all. The red curve represents the enzyme with an allosteric inhibitor, and the green curve represents the enzyme with an allosteric activator. And in this example, activators and inhibitors affect VO by either increasing or decreasing KM since the V max values seem to be pretty close between the three curves. So an activator here might be decreasing KM. Now, in this next example, we have the same three colored curves, but instead of KM changing significantly, the regulators seem to be changing V max. With the activator increasing the V max value. So, now that we've talked about activators and inhibitors, let's introduce the idea of the feedback loop. And, the basic idea is that a feedback loop is when you have downstream products regulating upstream reactions. And I understand this can be a mouthful, so let me show you this little reaction sequence, where we have A forming B through reaction one, and B forming C through reaction two, and so on and so on. Now let's say that molecule F acted as an activator for the ezyme powering reaction one. So it had a positive effect on enzyme one's activity. Now we would call this a positive feedback loop since molecule F increases the rate of reaction one, which then causes even more F to be made, since we've increased the increase the rate of formation of molecule F. Now, let's say that molecule F had a negative effect on enzyme one, we would call this a negative feedback loop since molecule F decreases the rate of reaction one, which leads to a decrease in the rate of formation in molecule F. So, let's look at an example of a feedback loop just to really drive home the point if you're still confused. Now, phosphofructokianase is an enzyme involved in glycolysis, and it catalyzes the conversion of fructose six phosphate and ATP to form fructose one six bisphosphate and ADP. Now, remember that glycolysis is a metabolic process that cells use to generate ATP. So, here, our molecule F, or downstream regulator from the last example, is ATP. and it turns out that ATP is an allosteric inhibitor of phosphofructokianase. And this makes sense because if ATP is at a high level, it's like the cell saying "We have ATP and we don't really need any more. "And we don't need phosphofructokianase "to push glycolysis along." So this would be a good example of a negative feedback loop. Since making ATP slows down glycolysis, and thus slows down the rate of ATP production. Now, because ATP is both an allosteric regulator and a substrate for phosphofructokianase, we can call it a homotropic inhibitor, which is a new term, and we call it a homotropic inhibitor because the substrate and the regulator are the same molecule. Now AMP, which is used up ATP, is an activator for phosphofructokianase, and this also makes sense because if AMP levels are high, then ATP levels are probably low. And it's like the cell saying "We need ATP." So we do need phosphofructokianase to push glycolysis along. Now, since AMP is a regulating molecule but not an active site substrate for phosphofructokianase it would be considered a heterotropic activator since the substrate and regulator are different. Now, the final point I want to make is that specific reactions make excellent control points for long, multistep processes. And remember that glycolysis is a ten step sequence. So why is there so much regulation going on for this one step? Well, this reaction in particular has a very negative delta G, and it's actually negative 4.5 kCal per mol. And that means that it's not easily reversed since there'll be a big release of energy from the reaction, and this makes THIS step of glycolysis an excellent control point for ALL ten steps together, since it's more or less a one way reaction. So, what did we learn? Well, first, we learned about the concept of allostery, and how regulatory molecules bind to allosteric sites instead of active sites. Second, we learned that these allosteric regulators influence an enzyme's kinetics by increasing KM or V max, and third we learned about what a feedback loop is, and how in long, multistep processes like glycolysis, the best control points are highly committing steps, the ones with very negative delta G values. |
32 | Biomolecules | Enzyme Kinetics | Non-enzymatic protein function | So we're going to be talking about nonenzymatic protein function. And before we get into what exactly that means, let's just say we are playing a game of Scattergories. And the next category that comes up is protein, and you have to list all the different types of protein that you know. And so you're like, oh, I totally got this. We've got beef and pork and chicken. Right? Those are different types of protein, and that's true. But what about the biochemistry type of protein, as in the large biomolecules that are made up of amino acids? What are the different types of proteins then? And so that's a little bit tougher. And so that's the point of what we're going to be talking about today is to talk about all the different types of proteins and the different functions that they perform. And so the key to understanding proteins as we go forward is understanding one unique characteristic about proteins. And that unique characteristic is that they can bind various biomolecules, and they bind specifically and tightly. And so just keep that concept in mind as we're talking about all the different functions of proteins, and that will help you understand how they are able to perform the vast array of functions that they do. So I like to think of proteins as being in one of two main classes. There are the enzymatic proteins, so enzymes, and then the nonenzymatic proteins, or we'll just call them the nonenzymes. And so let's back up just a minute. What exactly are enzymes? What do they do? Enzymes are, in a nutshell, little chemical reaction machines. They can catalyze all sorts of chemical reactions, so they catalyze reactions that help to sustain life. And so they are really the workhorses of the cell, helping to build up and break down things as needed. And by acting as catalyst, enzymes can help to accelerate the rate and specificity of these chemical reactions. And one good example of this that you're probably familiar with already is DNA polymerase, which catalyzes the synthesis of new strands of DNA. And probably even more familiar to you are the enzymes in your saliva. So every time you eat, there is one enzyme in particular called amylase, which is responsible for breaking down starch into sugars. So amylase is another example of a protein that has enzymatic function. And just notice for a second that both of these enzymes end in ASE, ase. And so in general, if you see a protein and it has this sort of ending, you should be thinking to yourself, oh, I bet this is an enzyme, an enzymatic protein. So that's a good rule of thumb. Now, nonenzymatic proteins, or nonenzymes, are all of those proteins that carry out functions that require the capacity to bind, but not necessarily to catalyze a reaction. And so what are some examples that we'll be talking about of nonenzymatic protein function? Well, there are proteins that function as receptors or ion channels in a cell membrane, and we'll talk more about that. And then there are proteins that are transport proteins. There also motor proteins. And then a special class of proteins that function as an integral part of the immune system, and those are called antibodies. And so we'll go through each of these examples of nonenzymatic protein function one by one. So let's give ourselves a little bit more room to talk about those. Now, let me quickly interject that it's important to realize that not all proteins are always either an enzyme or a nonenzyme. Oftentimes, they have characteristics of both, and so just keep that in mind as I'm going through and highlighting the nonenzymatic properties in this video. OK, so starting with receptors and ion channels. There are certain proteins that exist in the membrane of a cell and function as either receptors or ion channels. Now, receptors are proteins that receive, or bind, a signaling molecule. So let's draw a cell here. We're going to draw the membrane bilayer here. And so here would be my exterior of the cell, and here's the interior of the cell. And within this membrane bilayer, you can find a receptor protein. So we'll draw that here. And this receptor protein will bind a signaling molecule, also known as a ligand, which then induces some sort of chemical response in the cell. So one example of a receptor protein and ligand pair is an insulin receptor and insulin. So let's say this ligand is insulin, and this is the insulin receptor. Now, insulin is a hormone that's released by the pancreas in response to an increase in blood glucose levels. So let's say there's extra glucose, because you just ate a piece of pizza or something. So let's say there's an increase in glucose around, and then insulin is going to be released by the pancreas in response to this increase in blood glucose. And then once it's released, it binds to its corresponding receptor on certain cells, which leads to a cascade of signals within the cell. And this allows it to then absorb this excess glucose into the cell. And then likewise, you can also have an ion channel that also spans the membrane bilayer. So let's extend this lipid membrane bilayer, and then we'll draw an ion channel protein here. And so likewise, this protein spans the entire bilayer of the cell, and it acts as a pore or a channel through which certain ions, say calcium, can enter or exit the cell. So it can come in and come out through the same channel. So next up are the transport proteins, and now these proteins are responsible for binding small molecules and transporting them to other locations in a multicellular organism like humans. And the trick with these proteins is that they have to have a high affinity for their ligand when the ligand is present in high concentration. So at high concentration of a ligand, you have high affinity of the protein for that ligand, and at low concentration, you have low affinity. And a great example of this is hemoglobin. So hemoglobin is present in red blood cells, and it picks up oxygen in the lungs so here are my lungs, it's high in oxygen in your lungs and then delivers this oxygen to tissues. And we'll draw a tissue here, say it's muscle tissue, where its present in low concentrations. And so this is a great example of a transport protein at work. So next up are the motor proteins, which include myosin, kinesin, and dynein, all of which are capable of generating great forces. And these proteins are really crucial for cellular motility. And myosin specifically is a protein responsible for generating the forces exerted by contracting muscles. So every time you flex your bicep like so, your myosin protein in your muscles are contracting and generating that force. Now, kinesin and dynein are motor proteins that are responsible for intracellular transport. And then dynein in particular also plays a role in the motility of cilia, which are these little extensions of a cell that project out. And mutations in a particular dynein protein can lead to a rare disease called primary ciliary dyskinesia. So in primary ciliary dyskinesia, you can see there's some sort of dyskinesia or problem in movement for the cilia. And mutations in a particular dynein protein lead to this rare disease in which the action of the cilia of the cells lining the respiratory tract fail to function. And this leads to a decrease in mucus clearance from the lungs, and therefore an increase susceptibility to chronic infections like pneumonia and bronchitis. So as you can see, these motor proteins are really important. And then finally, our last class of nonenzymatic proteins that we'll be talking about are the antibodies of the immune system. Now, antibodies are protein components of the adaptive immune system whose main function is to find foreign antigens and target them for destruction. So in this case, the antigen, which comes from any foreign substance, say a virus or something like that, is the antibodies ligand. So here's an example of an antibody, and then here is the antigen. And the antigen is really just the antibodies particular ligand. So you can think of antibodies as being like little red flags for the body's immune system letting us know that hey, this thing is not supposed to be here. We need to get rid of it somehow. And it's important to know that an antibody's affinity for its target antigen is extraordinarily high. So the affinity is strong, really high. And so there you have it. You can see all the different types of nonenzymatic roles or functions that proteins can play, either as receptors or ion channels, as transport proteins, motor proteins, and then highly specific antibodies in our immune system. |
33 | Biomolecules | Enzyme Kinetics | Covalent modifications to enzymes | So today we're gonna learn about covalent modifications to enzymes. But first, let's review the idea that enzymes make reactions go faster. And looking at a reaction coordinate diagram you notice that enzymes do this by lowering the reaction's activation energy. Also, before we talk about covalently modified enzymes, I want to remind you that not all enzymes are proteins. And often, when we think of enzymes we think of proteins, which are amino acid polymers with primary, secondary, tertiary, and quadrinary structures. But there are also many different kinds of enzymes that aren't proteins. Inorganic metals, like magnesium, or small organic molecules, like flavin, can also act as enzymes. But for the purposes of this discussion we're going to focus on the proteins. And to be clear, when we say covalent modifications, we refer to modifications to a protein that involve forming or breaking covalent bonds. Now there are tons of different covalent modifications that we can observe. So I'm only gonna touch on a select few to get the point across. And the first category of covalent modifications I want to talk about are small posttranslational modifications. Now, when I say translation, I'm referring to the process of translation where amino acid polymers are synthesized. And when I say posttranslation, I refer to events that take place after that initial synthesis. Now when I say small, all I'm referring to are modifications that involve small functional groups being added or removed from an enzyme. And again, there are many different types of these but I'm just gonna touch on three. So methylation is a modification of a protein that involves the addition of a methyl group, or CH3, to a protein. Acetylation involves the addition of an acetyl group. And glycosylation involves the addition of a sugar molecule. And these are just three examples of a huge list. And these modifications, although small, can have pretty significant impacts on protein as a whole. And to discuss this, I want to mention the example of acetylation of a lysine residue on a protein. So as you many know, lysine is an amino acid that has an extra amino group on its side chain that can act as a base and carry a positive charge. If we were to acetylate this lysine residue and add an acetyl group to the amino and nitrogen, which is a covalent modification, the electron withdrawing effect of the acetyl group will prevent that nitrogen from carrying a positive charge, and modify the behavior of that amino acid. The loss of that positive charge can change a few properties of the amino acid, including changes to the lysine's acidity and basicity, since it can no longer exchange protons, as well. And it will also influence lysines electrostatic interactions with other charged molecules, since it's lost that positive charge. So even a small modification, like the addition of a cell group, can have significant impacts on the protein overall. Moving on, I want to discuss another way in which covalent modifications of enzymes is relevant. And that's in reference to zymogens. Now a zymogen is an inactive form of an enzyme that requires a covalent modification in order to become active. And a big example of these zymogens in biology are the digestive enzymes of the pancreas releases so that you can digest food. One of the enzymes of the pancreas releases is called trypsinogen, which is a zymogen as indicated by the ogen suffix. Now this is an inactive form of a chrodeus enzyme that is shipped to the intestine. And once in the intestine, it's covalently modified by an enzyme called enterokinase which converts it to its active form trypsin. Now this is to prevent trypsin from breaking down proteins that we need in the pancreas since it's inactive at that point as trypsinogen. And only allows it to break proteins down in the intestine after it's encountered enterokinase. Notice how you can distinguish zymogens from their active form by their name, zymogens have ogen added to the end of them. Now the last example of covalently modified enzymes that I want to discuss is the subject of suicide inhibition. Now, when we think of enzymatic inhibition we usually think of competetive, noncompetetitve, and uncompetetive inhibitors which follows certain patterns in terms of their effects on enzyme kinetics. But there's another type of inhibitor that's a little different, and this is the suicide inhibitor. Suicide inhibitors covalently bind the enzyme and prevent it from catalyzing reactions. And what's interesting is that since these inhibitors form covalent linkages to the proteins, they rarely unbind, which is why we call them suicide inhibitors. Since after they bind, that's it for them. And this is what distinguishes this type of inhibitor from the other three that you might be familiar with. So, what did we learn? Well, we talked about three very different things today that all have to do with covalent modifications to enzymes. First, we talked about small posttranslational modifications, like methylation, acetylation, and glycosylation. Second, we discussed zymogens, inactive proteins that require covalent modification to become active. And finally we talked about suicide inhibitors, which are enzyme inhibitors that permanently bind their target. |
34 | Biomolecules | DNA | DNA | As long as human beings have been around I could imagine that they have noticed that offspring tend to have traits in common with the parent. For example, someone might have told you, "Hey, you walk kind of like your dad," or, "Your smile is kind of like your mom," or, "Your eyes are like one of your uncles "or your grandparents." And so there's always been this notion of inherited traits. But it wasn't until the 1800 that that started to be studied in a more scientific way with Gregor Mendel the father of genetics. But even then, even Mendel who was starting to understand the mechanisms or he was trying to understand how inheritance happened, then you even could start to breed certain types of things. Even he didn't know exactly what was the molecular basis for inheritance. And the answer to that question wasn't figured out until fairly recent times, until the mid 20th century. Not until the structure of DNA was established by Watson and Crick and their work was based on the work of many others especially folks like Rosalind Franklin who essentially provided the bulk of the data for Watson and Crick's work, Maurice Wilkins and many, many, many other folks. But it's really the structure of DNA that made people say, "Hey, that looks like "the molecule that's storing the information." Just to be clear, DNA wasn't discovered in 1953. DNA was discovered in the mid 1800s. It was this kind of this molecule that was inside of nuclei of cells. And for some time people said, "Maybe this could be a molecular basis of inheritance." You could imagine what you would need to be a molecular basis of inheritance. It would have to be a molecule or a series of molecules that could contain information, that could be replicated, that could be expressed in some way. But it wasn't until 1953 wherein this double helix structure of DNA was established. The people said, "Hey, this looks like our molecule." So first, let's just talk about the structure here and then actually we'll talk about where this name, DNA, deoxyribonucleic acid comes from. And then we'll talk a little bit about why this structure lends itself well to something that stores information, that can replicate its information and that could express its information. We might go in depth on the expression of information in future videos. So this structure right over here and this is a visual depiction of a DNA molecule. You can view this as kind of a twisted ladder. It has these two, I guess you could say sides of the ladder that are twister. That is one side right over there and then it is another side. There is another side right over here. And in between those two sides or connecting those two sides of that twisted ladder you have these rungs. And these rungs are actually where the information, the genetic information is I guess you could say stored in some way. Because these rungs it's a sequence of different bases. And when I say bases, you're gonna say wait. This says acid, why are you saying bases right over here? Well, the word deoxyribonucleic acid comes from the fact that this backbone is made up of a combination of sugar and phosphate. And the sugar that makes up the backbone is deoxyribose. So that's essentially the D in DNA. And then the phosphate group is acidic and that's now where you get the acid part of it. And nucleic is, hey this was found in nuclei of cells. It is nucleic acid. Deoxyribonucleic acid. It is actually mildly acidic all in total but for every acid it actually also has a base, and those bases form the rung of the ladders. And actually each rung is a pair of bases and as I said, that's where the information is actually stored. Well what am I talking about? Well let me talk about the four different bases that make up the rungs of a DNA molecule. So, you have adenine. Adenine. And so for example, this part right over here. This section of that rung might be adenine. Maybe this right over here is adenine. This right over here. Remember, each of these rungs are made up by it's a pair of bases. And that might be adenine. Maybe this is adenine and I could stop there, I mean I'll do a little more adenine. Maybe that's adenine right over there. And adenine always pairs with the base thymine. So let me write that down. So adenine pairs with thymine. Thymine. So, if that's an adenine there then this is going to be a thymine. If this is an adenine then this is going to be a thymine. Or if I drew the thymine first, well say, okay it's gonna pair with the adenine. So this is going to be a thymine right over here. This is going to be a thymine. If I were to draw this, this would be a thymine right over here. Now the other two bases, you have cytosine which pairs with guanine or guanine that pairs with cytosine. So guanine and we're not gonna go into the molecular structure of these bases just yet, although these are good names to know because they show up a lot and they really form kind of the code, your genetic code. Guanine. Guanine pairs with cytosine. Guanine and cytosine. Cytosine. So actually if this is, let's say there's some cytosine there, let's say cytosine right over here. Maybe this is a cytosine, maybe this is cytosine, maybe this is cytosine, this is cytosine and maybe this is cytosine. Then it always pairs with the guanine. So, let's see, this is guanine then and this will be guanine. This is guanine, this is guanine. I actually didn't draw stuff here. This is guanine, I didn't say what these could be but these would be maybe the pairs of they could be adeninethymine pairs and it could be adenine on either side or the thymine on either side, and they could be made of guaninecytosine pairs where the guanine or the cytosine is on the other side. Actually just to make it a little bit more complete let me just color in the rungs here as best as I can. So those are guanines so they're gonna pair with cytosine. Pair with cytosine, pair with cytosine. When you straw in this way you might start to see how this is essentially a code, the order of which the bases are... I guess the order in which we have these or the sequence of these bases essentially in code the information that make you, you, and you could be. Well how much of it is nature versus nurture and when people say nature, you know, it's literally genetic, and that's an ongoing debate, an ongoing debate but it does code for things like your hair color. When you see that your smile is similar to your parents it is because that information to a large degree is encoded genetically. It affects a lot of what makes you you and actually not even just within a species but also across species. Humans have more genetic material in common with other humans than they do with say a plant. But all living creatures as we know them have genetic information. This is the basis by which they are passing down their actual traits. Now you might be saying well, how much genetic information does a human being have? And the number will either disappoint you or you might find it mindboggling. The human genome and every species has a different number of base pairs to large degree correlated with how complex they are although not always. But the human genome has 6,000,000. Sorry, not 6,000,000, 6,000,000,000. 6,000,000 would be disappointing, even billion might be disappointing. 6,000,000,000 base pairs. 6,000,000,000. 6,000,000,000 base pairs. And when you have your full complement of chromosomes and this is in most of the cells in your body and outside of your sex cells, the sperm or the egg cells. This is going to be spread over 46 chromosomes. 46 chromosomes or I guess you could say 23 pair of chromosomes. If you divide 6,000,000,000 by 46 you get a little over on average 100,000,000. I think it's a 100 and something million base pairs per chromosome. And some chromosomes are longer, actually the longest are over 200,000,000 and some might be shorter. That's just on average. Now this number might to some of you might be exciting. You're like, "I thought I was a simple creature. "I didn't know I was this complex." 6,000,000,000, that's a lot of base pairs. That feels like a lot of information. For others of you it might not feel so great. You might say, "Hey, wait I could store "this much information on a modern thumb drive "or on a hard disk. "I thought I was more unique than that." And of course we all are special and unique. You're gonna say 6,000,000,000 base pairs. I thought I was, you know, I was infinitely complex and whatever else. There's some arguments for that along some other directions, but this is the approximate length I guess you could say or the approximate size of the actual human genome. And when we talk about chromosomes and we'll talk about chromosomes in much more depth, imagine taking this zoomed in thing that you have right over here and you know, over here, I don't know how many we have, Like one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. We have about 20 base pairs depicted here. Imagine if you had about 200,000,000 of these base pairs and then you were to take this thing and you were to kind of coil it up into that thing is a chromosome. It is a chromosome and you're saying, "Wait, "I have that much information in "most of the cells of my body. "This thing must be incredible compact." And if you said that I would say, "Yes, you are correct." This, the radius, the radius of the DNA molecule is on the order of one nanometer. One nanometer which is a billionth of a meter. So you can start to assess kind of the scale of this thing. This is a very dense way to actually store information. But just to have an appreciation of and you might have seen it when I was coloring in on why the structure lends itself to being able to replicate the information or even to be able to translate or express the information. Let's think about if you were to take this ladder and you were to just kind of split all the base pairs. So, you just have 1/2 of them. So you essentially have half of the ladder. And so if you only have half of the ladder, you're able to construct the other half of the ladder. Let's take an example, let's say and I'll just use the first letter to abbreviate for each of these bases. Let's say you have some... So let's say this is one of the, this is the sugar phosphate backbone right over here. So this could be one of the sides. Let's say there's some adenine. Actually we do in the right color. So you got some adenine, adenine. Maybe some adenine right over here and maybe there's an adenine there. And maybe you have some thymine, thymine, maybe thymine right over here and then you have some guanine, guanine, guanine. And then let's say you have some cytosine and you have some cytosine. So with just half of this ladder I guess you could say, you're able to construct the other half, and this is actually how DNA replicates. This ladder splits and then each of those two halves of that ladder are able to construct versions of the other half, or versions of the other half are able to constructed on top of that, on top of that half. So how does that happen? Well, it's based on how these bases pair. Adenine always pairs with thymine if we're talking about DNA. So if you have an A there, you're gonna have a T on this end, T on this end. T's right all over here, T right over there. If you have a T on that end you're gonna have an A right over there. A, A. If you have a G, a guanine on this side, you're gonna have a cytosine on the other side. Cytosine, cytosine, cytosine. And if you have a cytosine you're gonna have a guanine on the other side. Hopefully that gives you an appreciation of how DNA can replicate itself. And as we'll see also how this information can be translated to other forms of either related molecules but eventually to proteins. And just to kind of round out this video, to get a real visual sense what the DNA molecule looks like or I guess a different visual depiction from this. I found this animated gif that, you know, if you haven't fully digested what a double helix looks like, this is it. And you see here, you see your sugar phosphate bases here. You see kind of the sugars and phosphate, the sugars and the phosphates alternating along this backbone, and then the rungs of the ladder are these base pairs. So this is one of the bases, that's the corresponding, that's this corresponding, I guess you can say partner. And you can see that along all the way up and down in this molecule. Very exciting. |
35 | Biomolecules | DNA | Molecular structure of DNA | We already have an overview video of DNA and I encourage you to watch that first. What I want to do in this video is dig a little bit deeper. Actually get into the molecular structure of DNA. This is a starting point. Let's just remind ourselves what DNA stands for. I'm gonna write the different parts of the word in different colors. It stands for deoxy. Deoxyribonucleic. Ribonucleic. Ribonucleic acid. Ribonucleic acid. So I'm just gonna put this on the side and now let's actually look at the molecular structure and how it relates to this actual name, deoxyribonucleic acid. DNA is just a junction for nucleic acid and it's the term nucleic that comes from the fact that it's found in the nucleus. It's found in the nucleus of eukaryotes. That's where the nucleic comes from and we'll talk about in a second why it's called an acid but I'll wait on that. Now each DNA molecule is made up of a chain of what we call nucleotides. What we call nucleotides. It's made up of nucleo, nucleo, nucleotides. What does a nucleotide look like? Well, what I have right over here is I have two strands, I've zoomed two strands of DNA or I've zoomed in two strands of DNA. You could view this side right over here as one of the, I guess you can say the backbones of one side of the ladder. This is the other side of the ladder and then each of these bridges, and I will talk about what molecules these are. These are kind of the rungs of the ladder. A nucleotide, let me separate off the nucleotide. A nucleotide would... What I am cordoning off, what I am cordoning off right over here could be considered, could be considered a nucleotide. That's one nucleotide and then it's connected to another. It's connected to another nucleotide. Another nucleotide right over here. On the right hand side we have a nucleotide, we have a nucleotide right over there and then, actually I want to do it, let me do it slightly different. We have a nucleotide right over here on the right side and then right below that we have another. We have another nucleotide. We have another nucleotide. Depicted here, we essentially have four nucleotides. These two are on this left side of the ladder, these two are on the right side of the ladder. Now let's think about the different pieces of that nucleotide. The one thing that might jump out at you is we have these phosphate groups. This is a phosphate group right over here. This is a phosphate group right over here. Each of these nucleotides have a phosphate group. This is a phosphate group over here and this is a phosphate group over here. Now the phosphate groups are actually what make DNA or actually what make nucleic acid an acid. You might say, wait, wait. The way you've drawn it Sal, you have a negative charge. Something with a negative charge would attract protons, it would sap up protons. How can you call this an acid? This actually looks more basic. The reason why its DNA is typically drawn with these negative charges here is that it's so acidic and that if you put it in into a neutral solution, it's actually going to lose its hydrogens. Actually the DNA if we actually want to be formal about it, the DNA molecules would actually have its phosphates protonated like this but it so badly wants to lose these hydrogen protons so it typically would be, let me draw it like this. Let me get rid of the negative charge just on this one. Whoops. Just on this phosphate group over here. If you get rid of the negative charge and if this was bounded, this is bonded to a hydrogen. This so badly wants to grab these electrons. These oxygen can grab these electrons and then these hydrogen will just be grabbed by another water molecule or something so the proton will be let go. That's why we call it an acid. If it wasn't in a solution it would have the hydrogens but it would be very acidic as soon as you put it into a neutral solution it's going to lose those hydrogens. The phosphate groups are what make it, are what make it an acid but it's confusing sometimes because usually when you see it depicted, you see it with these negative charges and that's because it has already lost its hydrogen proton. You're actually depicting the conjugate base here but that's where it gets its acidic name from because it starts protonated or it gets in this acid form, it's protonated but it readily loses it. And so that's why it has its, that's where it gets the name acid form from. Each of these nucleotides they have a phosphate group. Now the next thing you might notice, the next thing you might notice is. The next thing you might notice is this group right over here. It is a cycle, it is a ring and it looks an awful lot like a sugar and that's because it is a sugar. This sugar is based on, it's a fivecarbon sugar. What I have depicted here, this sugar, this is ribose. This sugar right over here is ribose. This is when it's just as a straight chain and like many sugars, it can take a cyclical form. Actually it can take many different cyclical forms but the one that's most typically described is when you have that. Let me number the carbons because carbon numbering is important when we talk about DNA. But if we start carbonyl group right over here we call that the one carbon or the one prime carbon. One prime, two prime, three prime, four prime and five prime. That's the five prime carbon. You form the cyclical form of ribose as if you have the oxygen. You have the oxygen right over here on the four prime carbon. It uses one of its lone pairs. It uses one of its lone pairs to form a bond. To form a bond with the one prime. With the one prime carbon and I drew it that way because it kind of does bend. The whole molecule's going to have to bend that way to form this structure. And then when it forms that bond the carbon can let go of one of these double bonds and then that can, then the oxygen, the oxygen can use that. The oxygen can use those electrons to go grab a hydrogen proton from some place. To nab on to a hydrogen proton. When it does that you're in this form and this form, just to be clear of what we're talking about, this is the one prime carbon. One prime, two prime, three prime, four prime and five prime carbon. Where we see this bond, this is the one prime carbon. it was part of a carbonyl. Now it lets go of one of those double bonds so that this oxygen can form a bond with a hydrogen proton. It let go of a double bond there so that this could form a bond with a hydrogen proton. This hydrogen proton is that hydrogen proton right over there and this green bond that gets formed between the four prime carbon and or between the oxygen that's attached to the four prime carbon and the one prime carbon, that's this. That's this bond right over here. This oxygen is that oxygen right there. Notice, this oxygen is bound to the four prime carbon and now it's also bound to the one prime carbon. It was also attached to a hydrogen. It was also attached to a hydrogen so that hydrogen is there but then that can get nabbed up by another passing water molecule to become hydronium so it can get lost. It grabs up a hydrogen proton right over here and so it can lose a hydrogen proton right there. It's not adding or losing in that net. You form this cyclical form and the cyclical form right over here is very close to what we see in a DNA molecule. It's actually what we would see in an RNA molecule, in a ribonucleic acid. And so what do we think we're talking about when we say deoxyribonucleic acid. Well, you can start with you have a ribose here but if we got rid of one of the oxygen groups and in particular one of... Well, actually if we just got rid of one of the oxygens we replace a hydroxyl with just a hydrogen, well then you're gonna have deoxyribose and you see that over here. This fivemember ring, you have four carbons right over here. it looks just like this. The hydrogens are implicit to the carbons, we've seen this multiple time. The carbons are at where these lines intersect or I guess at the edges or maybe and also where these lines end right over there. But you see this does not have an... This molecule if we compare these two molecules, if we compare these two molecules over here, we see that this guy has an OH, and this guy implicitly just has... This has an OH and an H. This guy implicitly has just two hydrogens over here. He's missing an oxygen. This is deoxyribose. Deoxyribose. Deoxyribose doesn't have this oxygen. It does not have the oxygen on the two prime carbon. So this if you get rid of that, this is deoxyribose. So let me circle that. This thing right over here, this thing right over here, that is deoxyribose. Deoxy or it's based on deoxyribose I guess before it bonded to these other constituents. You could consider this deoxyribose. That's where the deoxyribo comes from and then the last piece of it, the last piece of it is this chunk right over here. These we call nitrogenous bases. Nitrogenous. Nitrogenous. Nitrogenous bases. You could see we have different types of nitrogenous bases. This is a nitrogenous base. This right over here is a different nitrogenous base. This right over here is another different nitrogenous base. Notice, this one only has one ring, this one has one ring, this one has two rings. This one over here has two rings and we have different names for these nitrogenous bases. The ones with two rings, the general categorization we call them purines. Nitrogenous bases if you have two rings, if you have two rings we call them purines. That's a general classification term. Let me make sure, purines. If you have one ring. Anyway, I'll just write this way. One ring. One ring, we call these pyrimidines. Pyrimidine. Pyrimidines. We call these pyrimidines. These particular, these two on the right, these two purines, this one up here this is adenine, and we talk about how they pair in the overview video on DNA. This one right over here is adenine, this nitrogenous base. This one over here is guanine. That is guanine. And then over here, over here, this single ring nitrogenous base which makes it a pyrimidine, this is thymine. This right over here is thymine. This is thymine and then last but not least if we're talking about DNA, when we go into RNA, we're also gonna talk about uracil. But when we talk about DNA this one over here is cytosine. Cytosine. You could see the way it's structured. The thymine is attracted to adenine. It bonds with adenine and cytosine bonds with guanine. How are they bonding? Well, the way that these nitrogenous bases form the rungs of the ladder, how they want they're drawn to each other, this is our good old friend hydrogen bonds. This all comes out of the fact, that nitrogen is quite electronegative. When nitrogen is bound to a hydrogen you're going to have a partially negative charge at the nitrogen. Let me do this in green. You're going to have a partial negative charge at the nitrogen and a partially positive charge at the hydrogen. And then oxygen we've always talked about as being electronegative so it has a partial negative charge. The partial negative charge of this oxygen is going to be attracted to the partial positive charge of this hydrogen, and so you're going to have a hydrogen bond. That's then going to happen between this hydrogen which is going... Its electrons are being hogged by this nitrogen and this nitrogen with who, which itself hogs electrons. That forms a hydrogen bond. And then down here you have a hydrogen that has a partially positive charge because its electrons are being hogged. And then you have this oxygen with a partially negative charge, they're going to be attracted to each other. That's a hydrogen bond. Same thing between this nitrogen and that hydrogen, and same thing between this oxygen and that hydrogen. That's why cytosine and guanine pair up and that's why thymine and adenine pair up, and we talk about that as well in the overview video of DNA. |
36 | Biomolecules | DNA | Antiparallel structure of DNA strands | In the video on the molecular structure of DNA we saw that DNA is typically made up of two strands where the backbone of each of the strands is made up of phosphate alternating between a Do some different colors. A phosphate group and then you have a sugar. You have a phosphate group. And then you have a sugar. And then you have a phosphate group. And then you have a sugar. And so I could draw the strand something like this. So phosphate and then we have a sugar. Oops, let me just draw all the phosphates ahead of time. So you have the phosphates on that end and then you have the sugars. And you see the same thing on the other strand as well. Where we have phosphate with a sugar then another phosphate then a sugar then another phosphate. Let me circle the sugars as well. So we have a sugar there and then you have the sugar there as well. So on the other strand it's also going to look like this. So let me draw the phosphates. I'm just abstracting them now. So the phosphate and then you have the sugars in between the phosphates. And what links them, you can think of them as the rungs on the ladder. These are the complementary nitrogenous bases. And the reason why we call them nitrogenous bases, I actually forgot to talk about it in the last videos, is that these nitrogens are really electronegative and they can take up more hydrogen protons. They have an extra lone pair. The nitrogens have an extra lone pair that can be used up under the right conditions to potentially sop up more hydrogen protons. Now, a lot of people ask, "Well, if you have these nitrogenous bases here, "why is DNA called an acid?" Why is it called an acid?" Well the first thing is that the basic properties of the nitrogenous base are offset to a good degree based on the fact that they're able to hydrogen bond with each other. And that's what actually forms the rungs of the ladder when these complimentary nitrogenous bases form these hydrogen bonds with each other. But even more, the reason why we call it an acid is the phosphate groups, when they're protonated, are acids. Now the reason why we tend to draw them deprotonated is they're so acidic that if you put them in a neutral solution, they're going to be deprotonated. So this is the form that you're more likely to find it in the nucleus of an actual cell. Once it's actually already deprotonated. But in general, phosphate groups are considered acidic. And if I were to draw kind of a more pure phosphate group, and I talked about this already in the last video, I would have it protonated and so I wouldn't draw that negative charge like that. So that's just a review of last time. Since I already started abstracting it, let's abstract further. So let's draw the nitrogenous bases a little bit. So I have thymine here. And I will do thymine in this green color. So this right over there is thymine. So this is attached to thymine. And the complementary nitrogenous base to thymine is adenine. Which I will do Let's see I'm running out of colors here. Let's see. Adenine. I'll do this in an orange color since it's got so many nitrogens on it. So actually should include that hydrogen right over there. So this right over here is adenine. Now they have these hydrogen bonds between them right over here. Because they have partially negative and positive charges on either end that are attracted to each other. And then we go to this rung, one rung below it. And what is going on? Well, let's see we have Now I really am running out of colors here. We have this nitrogenous base is cytosine. This nitrogenous base right over here is cytosine. This nitrogenous base here is cytosine. And it is paired up with guanine. It is paired up with guanine. I'll do guanine in this color. So it is paired up with guanine right over there. And we even saw this in the introductory video to DNA. Now you might say, "Oh look, these two strands "seem parallel to each other." And in some ways that is true. But there might be another interesting thing that you might have noticed, is the direction in which they are oriented. I guess is the best way to phrase it. And you especially see that when you focus in on the sugars. Notice the sugars over here, the deoxyriboses or the parts of the nucleotide that come from deoxyribose. You see the oxygens on the top of the ribose, on the top of these five member rings. The oxygen is on top. Well on this side, the oxygen is on the bottom. And so they're actually in different orientations. Here the oxygen is pointing up, here the oxygen is pointing down. And to get a little bit more concrete about that. We can number the carbons on the ribose to think about the directions and use those numbers of the carbons to describe the different directions. So let's number our carbons. So these are both ribose, we saw that in the molecular structure of DNA videos. When we're talking about DNA we're talking about deoxyribose. Instead of having a hydroxyl group on the number two carbon, it just has a hydrogen. So instead of having a hydroxyl group on the number two carbon, it just has a hydrogen. But let's actually number them. So this is the one prime carbon starting at the carbonyl group. Let me do that in a different color. So this is the one prime carbon. And I'm just numbering them starting at the carbonyl group. One prime. Two prime. Three prime. Four prime. Five prime. And then when you look at it as a ring, this was the one prime. This is the two prime. This is the three prime. This is the four prime. This is the five prime. Or if you were to number them on this diagram right over here, actually in the DNA molecule, this is the one prime. This is the two prime carbon. This is the three prime carbon. This is the four prime carbon. And this is the five prime carbon. And so one way to think about it is we'll go phosphate group and it's connected with what we call phosphodiester linkages. Phosphodiester linkages, that's what's essentially allowing these backbones to link up. But we're going from phosphate to five prime carbon and then through the sugar we go to the three prime carbon. And then we go to another phosphate. Then we go to the five prime carbon. Let me label that, this is the five prime carbon. Then we go to the three prime carbon. And that just comes straight out of just numbering these starting with the carbon that was a number one carbon. When it's straight chain form, it's part of the carbonyl group. But you see we're going from five. We go phosphate, five prime, three prime, phosphate, five prime, three prime, phosphate. So one way to describe the orientation is saying, "Hey, we're going in the direction "from five prime to three prime." So we could say that we're going from five prime to three prime, that way on the lefthand chain. And what are we doing on the right hand chain? Well, let's number them again. So this is the one prime carbon. Now this thing relative to this is upside down, it's inverted. So one prime. Two prime. Three prime. Four prime. Five prime. I could do it up here. One prime carbon. Two prime carbon. Three prime carbon. Four prime carbon. Five prime carbon. Here you're going from phosphate, three prime, five prime, phosphate, three prime, five prime, phosphate. So the way that the sugars are oriented if you're going from top to bottom the way we're looking here, you're going from three prime to five prime. So on the right hand side, it's three prime, five prime. And so if you wanted to draw an arrow from five prime to three prime, you could look at it like that. And so you could say these are parallel but since they are essentially pointing in different directions even though they are actually parallel, we would call this structure of DNA antiparallel. So this would be an anti... antiparallel structure of DNA. So these two strands, they're complementary. They're defined by each other. The thymine bonds with the adenine, the cytosine bonds with guanine. They are attracted to each other through these hydrogen bonds. But the two backbones, they're pointed in different directions. And now another interesting thing to think about, since we're talking about the molecular structure of DNA, is how do these things form? How did these things know to orient in this way? What plays part of that role is the fact that these phosphate groups are negative. So you think these things that have outright negative charge, they're gonna try to get as far away from each other as possible. And then when they just keep kind of orienting and getting far away from each other. And these are long. These are very, very, very, very long molecules. In the introductory video to DNA we talk about how long these chromosomes are, how many base pairs we actually have. And these are long molecules. So all of these phosphate groups on either strand, they want to get away from each other. And then these things want to get close to each other because of the hydrogen bonds. And so that's what helps form this actual ladder structure. So DNA, fascinating molecule, we could speak for days about it. It's actually mind blowing when you think about its implications for who we are. But hopefully this gives you a better sense of what it is molecularlaly. molecularlaly. I cannot say it. Molecularly. |
37 | Biomolecules | DNA | Telomeres and single copy DNA vs repetitive DNA | Here is a pair of chromosomes as they would appear during mitosis, and the ends of chromosomes are capped with an area known as telomeres, and telomeres are mainly found in eukaryotic chromosomes because usually prokaryotes just have one circular chromosome, so it doesn't have any ends. And what do telomeres do? Well, they protect chromosomes, or protect the ends of chromosomes from deterioration. Why would the ends of chromosomes deteriorate? So, the enzymes that replicate chromosomes are not able to get to the very, very end of the chromosome. So there's gonna be, they're gonna get to, let's say this area. So there's gonna be a small spot over here that's not replicated, and since the telomeres don't have any genes in them, it's not really harmful, it doesn't really matter. So, what would happen if there were no telomeres? Well, let's take a look at the other chromosome. If there were no telomeres, and let's say, the chromosome was only replicated 'til about here, there would be this area with useful genes that wouldn't be replicated, and that would be pretty problematic. So basically, telomeres act as a buffer zone, because they do not contain any important genes. Another thing that telomeres do is they prevent chromosomes from sticking to each other. If chromosomes stuck to each other, then a lot of the genes would be scrambled and genes wouldn't be where they're supposed to be and that would be pretty problematic. And here's actually a picture of human chromosomes where the telomeres are highlighted in this florescent, so you can see the telomeres over here. So you can see how at both ends of each chromatid, there are telomeres, and so what happens is that with each time the chromosomes replicate, the telomeres get a little bit shorter and shorter and shorter, so there's an enzyme known as telomerase and telomerase is able to lengthen telomeres and bring them back to their original length. So there are some cells that replicate a lot and they have a lot of telomerase. This cell can keep on replicating and replicating, but then there are other cells that do not have a lot of telomerase, and when telomeres are basically nonexistent anymore, because the chromosomes replicated many, many times, let's just get rid of the telomeres, so the chromosomes will actually not be able to replicate, and so the cell will not divide again, and it will kind of die. Now that we're talking about telomeres, I want to bring up a topic that's tangentially related, and that is single copy DNA and repetitive DNA. So, single copy DNA is when you have a DNA sequence, I'm just gonna make one up, let's say, A T C C, that basically does not repeat itself, so it might be flanked by other DNA sequences, as opposed to repetitive DNA, which is when you have a DNA sequence that keeps repeating itself, so you might have it A T C C and then again, A T C C, A T C C, et cetera. So what's the difference between single copy DNA and repetitive DNA? So here we have a spectrum. On the left we have single copy DNA, in the middle we have DNA that's somewhat repetitive, and on the right we have highly repetitive DNA. So, single copy DNA holds most of the organism's, there should be an apostrophe there, important genetic information, so basically most of the important genes are going to be single copy, so since the important genes are single copy DNA, single copy DNA is transcribed and translated and it has a low mutation rate, which is a good thing because of course, we don't want there to be mutations in the important genes. Repetitive DNA, or DNA that's somewhat repetitive is found, well at least in mammals and in insects, near the centromeres. If you recall, the centromeres are the center of the chromatid, or when you have chromatids that are duplicated, the chromatids are attached by the centromere, by the, that middle part in the chromosome, and they may contain genes that are transcribed and translated, but then there might also be parts of the repetitive DNA that don't contain genes, and those parts are not transcribed and translated, and repetitive DNA has a higher mutation rate than single copy DNA. Now let's take a look at DNA that's highly repetitive. So, it contains no genes, and because it contains no genes, it is not transcribed and not translated, and highly repetitive DNA has an even higher rate of mutation than DNA that's somewhat repetitive. So there's lots of highly repetitive DNA that we're not exactly sure what its purpose is. Scientists are currently trying to figure out what the purpose of this highly repetitive DNA is, but there are some sections of highly repetitive DNA that we do know what their purpose is. For example, telomeres. Telomeres are sections of highly repetitive DNA and as I've explained before, their purpose is to basically act as a buffer zone for the important part of the chromosome, and in fact, the DNA sequence that's repeated in telomeres is this right over here, G G T T A G, and in human chromosomes, the telomeres are made up of approximately 2,000 repeats of this DNA sequence, G G T T A G. |
38 | Biomolecules | DNA | Leading and lagging strands in DNA replication | Let's talk a little bit in more depth about how DNA actually copies itself, how it actually replicates, and we're gonna talk about the actual actors in the process. Now, as I talk about it, I'm gonna talk a lot about the 3' and 5' ends of the DNA molecule, and if that is completely unfamiliar to you, I encourage you to watch the video on the antiparallel structure of DNA. And I'll give a little bit of a quick review here, just in case you saw it but it was a little while ago. This is a zoomin of DNA, it's actually the zoomin from that video, and when we talk about the 5' and 3' ends, we're referring to what's happening on the riboses that formed part of this phosphate sugar backbone. So we have ribose right over here, fivecarbon sugar, and we can number the carbons; this is the 1' carbon, that's the 2' carbon, that's the 3' carbon, that's the 4' carbon, and that's the 5' carbon. So this side of the ladder, you could say, it is going in the ... it is going, let me draw a little line here, this is going in the 3' to 5' direction. So this end is 3' and then this end is 5'. It's going 3' to 5'. Notice three, this phosphate connects to the 3', then we go to the 5' connects to a phosphate, this connects to a 3', then it connects then we go to the 5' connects to a phosphate. Now on this end, as we said it's antiparallel. It's parallel, but it's oriented the other way. So this is the 3', this is the 5', this is the 3', this is the 5'. And so this is just what we're talking about when we talk about the antiparallel structure. These two backbones, these two strands are parallel to each other, but they're oriented in opposite directions. So this is the 3' end and this is the 5' end. And this is gonna be really important for understanding replication, because the DNA polymerase, the things that's adding more and more nucleotides to grow a DNA strand; it can only add nucleotides on the 3' end. So if we were talking about this right over here, we would only be able to add … We would only be able to add going that way. We wouldn't be able to add going … We wouldn't be able to add going that way. So one way to think about it is you can only add nucleotides on the 3' end or you can only extend … You can only extend DNA going from 5' to 3'. If you're only adding on the 3' end, then you're going from the 5' to the 3' direction. You can't go from the 3' to the 5' direction. You can't continue to add on the 5' side using polymerase. So what am I talking about with polymerase. Well let's look at this diagram right over here that really gives us an overview of all of the different actors. So here is just our of our DNA strand, and it's, you can imagine it's somewhat natural, in it's natural unreplicated form, and you could see we've labeled here the 3' and the 5' ends, and you could follow one of these backbones. This 3', if you follow it all the way over here, it goes, this is the corresponding 5' end. So this and this are the same strand, and this one, if you follow it along, if you go all the way over here, it's the same strand. So this is the 3' end, and 3' end of it and then this is the 5' end of it. Now the first thing, and we've talked about this in previous videos where we give an overview of replication, is the general idea is that the two sides of our helix, the two DNA, the doublehelix needs to get split, and then we can build another, we can build another side of the ladder on each of those two split ends. You could really view this as if this is a zipper, you unzip it and then you put new zippers on either end. But there's a lot of in reality, it is far more complex than just saying "Oh, let's open the zipper and put new zippers on it." It involves a whole bunch of enzymes and all sorts of things and even in this diagram, we're not showing all of the different actors, but we're showing you the primary actors, at least the ones that you'll hear discussed when people talk about DNA replication. So the first thing that needs to happen, right over here, it's all tightly, tightly wound. So let me write that, it is tightly, tightly wound. And it actually turns out, the more that we unwind it on one side, the more tightly wound it gets on this side. So in order for us to unzip the zipper, we need to have an enzyme that helps us unwind this tightly wound helix. And that enzyme is the topoisomerase. And the way that it actually works is it breaks up parts of the back bones temporarily, so that it can unwind and then they get back together, but the general highlevel idea is it unwinds it, so then the helicase enzyme, and the helicase really doesn't look like this little triangle that's cutting things. These things are actually far more fascinating if you were to actually see a the molecular structure of helicase. But what helicase is doing is it's breaking those hydrogen bonds between our … Between our nitrogenous bases, in this case it's an adenine here, this is a thymine and it would break that hydrogen bond between these two. So, first you unwind it, then the helicase, the topoisomerase unwinds it, then the helicase breaks them up, and then we actually think about these two strands differently, because as I mentioned, you can only add nucleotides going from the 5' to 3' direction. So this strand on the bottom right over here which we will call our leading strand, this one actually has a pretty straightforward, remember this is the 5' end right over here, so it can add, it can add going in that direction, it can add going in that direction right over here. This is the 5' to 3', so what needs to happen here is to start the process, you need an RNA primer and the character that puts an RNA primer, that is DNA primase. We'll talk a little bit more about these characters up here in the lagging strand, but they'll add an RNA, let me do this in a color you can see, an RNA primer will be added here, and then once there's a primer, then DNA polymerase can just start adding nucleotides, it can start adding nucleotides at the 3' end. And the reason why the leading strand has it pretty easy is this DNA polymerase right over here, this polymerase, and once again, they aren't these perfect rectangles as on this diagram. They're actually much more fascinating than that. You see the polymerase up there, you also see you one over here, polymerase. This polymerase can just, you can kind of think of it as following the opened zipper and then just keep adding, keep adding nucleotides at the 3' end. And so this one seems pretty straightforward. Now, you might say wouldn't it be easy if we could just add nucleotides at a 5' end, because then we could say well this is going from 3' to 5', well maybe that polymerase or different polymerase could just keep adding nucleotides like that, and then everything would be easy. Well, it turns out that that is not the case. you cannot add nucleotides at the 5' end, and let me be clear, this 3' right over here, this, I'm talking about this strand. This strand right over here, this, let me do this in another color, this strand right over here, this is the 3' end, this is the 5' end, and so you can't, you can't just keep adding nucleotides just like that, and so how does biology handle this? Well it handles this by adding primers right as this opening happens, it'll add primers, and this diagram shows the primer is just one nucleotide but a primer is typically several nucleotides, roughly 10 nucleotides. So it'll add roughly 10 RNA nucleotides right over here, and that's done by the DNA primase. So the DNA primase is going along the lagging, is going along this side, I can say the top strand, and it's adding, it's adding the RNA primer, which won't be just one nucleotide, it tends to be several of them, and then once you have that RNA primer, then the polymerase can add in the 5' to 3' direction, it can add on the 3' end. So then it can just start adding, it can just start adding DNA like that. And so you can imagine this process, it's kind of, you add the primase, put some primer here, and then you start building from the 5' to 3' direction. You start building just like that, and then you skip a little bit and then that happens again. So you end up with all these fragments of DNA and those fragments are called Okazaki fragments. So, it's a Okazaki fragments, and so what you have happening here on the lagging strand, you can think of it as, why is it called the lagging strand? Well you have to do it in this kind of … it feels like a suboptimal way where you have to keep creating these Okazaki fragments as you follow this opening, and so it lags, it's going to be a slower process, but then all of these strands can be put together using the DNA ligase. The DNA ligase; not only will the strands be put together, but then you also have the RNA being actually replaced with DNA and then when all is said done, you are going to have a strand of DNA being replicated, or being created right up here. So when it's all done, you're gonna have two double strands, one up here for on the lagging strand, and one down here on the leading strand. |
39 | Biomolecules | DNA | Transcription and mRNA processing | What we're going to do in this video is a little bit of a deep dive on transcription. And just as a bit of a review, we touch on it on the video on replication, transcription and translation. Transcription in everyday language just means to rewrite something or to rewrite some information in another form. And that's essentially what's happening here. Transcription is when we take the information encoded in the gene in DNA and encode essentially that same information in mRNA. So transcription we are going from DNA to messenger RNA, and we're gonna, in this video, focus on genes that code for proteins. So this first step is the transcription, the DNA to messenger RNA, and then in a future video we'll dig a little bit deeper into translation. We will translate that information into an actual protein. But these diagrams give a little bit of an overview of it. It's a little bit simpler in bacteria. You have the DNA just floating around in the cytosol, and so the transcription takes place. You start with that DNA, that protein coding gene in the DNA, and from that you code the messenger RNA, you see that in that purple color right over here, and then that messenger RNA can be involved with the ribosome, and that's the translation process to actually produce the polypeptide, to produce the protein. In eukaryotic cells, and we're going to get into a little bit more depth in this video, the transcription, the DNA to mRNA, that happens inside of the nucleus. There's essentially two steps here. You go from DNA to what we would call premRNA, let me write that down, premRNA, which is depicted right over there, and then it needs to be processed to turn into what we would call mRNA, which then can leave the nucleus to be translated into a protein. So now that we have that overview, let's dig a little bit deeper into this and understand the different actors and understand if we're talking about a eukaryotic cell what type of processing might actually go on. So right over here, we are going to start with the protein coding gene inside of the DNA, right over here, and the primary actor that's not the DNA or the mRNA here is going to be RNA polymerase. It's used to create a sequence that will become a nucleotide sequence, that will become the messenger RNA. So this RNA polymerase, it needs to know where to start. The way it knows where to start is it attaches to a sequence of the DNA known as a promoter. And every gene is going to have a promoter associated with it, especially if we're talking about eukaryotic cells. Sometimes you might have a promoter associated with a collection of genes as well. But in general, if you've got a gene, you're gonna have a promoter. That's how the RNA polymerase knows to attach right over there. Once it attaches, well then, it is able to separate the strands. It separates the strands, and it's pretty interesting, because when we went in deep into replication, you saw all of these actors, the helicase and whatever else, but this RNA polymerase complex is actually quite capable. Not only it separates the strand and then it's actually able to code for the RNA. It does that the same way that when we studied DNA polymerase, it does it in only one direction. It can only add more nucleotides on the three prime end. So it encodes from the five prime to the three prime direction. Notice this arrow here, we're extending it on the three prime end of the RNA. So as you can see here, when it does this, it's only encoding one side of... Or it's only interacting, I guess you could say, or coding complementary information to one side. But let's think about this a little bit. We could call the side that it is interacting with, you can call that the template strand because that side of the DNA is acting as the template for forming that RNA. But if you think about the information that that RNA is actually going to encode, well it's gonna contain the same information as the coding strand of DNA, as the other stand of DNA, because these nucleotides right over here, this nucleotide is going to be complementary to this one over here, just as this nucleotide was complementary to that one over there. And you can see it in a little bit more depth if we actually were to add the nucleotides. So this is the template strand. If you have a thymine, well on the RNA, you'd have the adenine. Look, on the coding strand of DNA, the one up here, you would also have an adenine. Essentially the coding strand and the RNA, essentially end up being the same sequence, but the one difference is that you won't find the thymine in the RNA, instead you'll find a similar nitrogenous base, and that is uracil. But uracil plays the role of thymine, so you're essentially coding the same information. So once again, this bottom strand is acting as a template, but it's going to be the resulting RNA that gets coded, is essentially going to have the same information that we had in the coding strand. Just to get an appreciation for what this looks like, I would even write, I'd put looks in quotations, I even did little quote things with my fingers when I said that, is that it's hard to really visualize what these things look like, but you can see here that the RNA polymerase complex, and this is for a specific organism, can be very, very complex and involved, and it's fascinating how these things interact. Every time you're studying biology and someone like me is going to give you these nice clean narratives of how these enzymes interact with the different macromolecules, like the DNA or the RNA, you should always remember this is amazing. These are these molecules interacting with each other, bouncing into each other. It's happening incredibly fast inside of the cell. You should be in awe of this. It's happening in all of your cells or as we speak. This is pretty incredible stuff. So the next thing you have to think about, this right over here, we are extending the RNA, well when does this thing actually stop? It stops once we... So this RNA polymerase is going to keep going on and then this blue, we've labeled this a terminator. So let me write. So this area is a terminator, and there's multiple ways that that signals to the RNA polymerase that "Hey, it's time to stop." More particularly, it somehow creates something structurally that the polymerase just lets go. One mechanism, that's depicted right over here is that the mRNA that's coded, this could happen in bacteria, is that the mRNA that's coded forms a hairpin. So it has to have the right complementary base pairs, base pairs right over here, to form this hairpin. This hairpin, along with the things around the hairpin, essentially make it, impair the polymerase to keep on going. So, the complex kind of changes a little bit. So, it let's go, or at least that's how people believe it. There's other forms of how the terminator can act. It might be sequences that parts of the polymerase complex recognize and it makes a conformation change so that the RNA polymerase lets go. If we're talking about a prokaryote, we're done. This would be our messenger RNA which then can go to a ribosome and then be translated into a protein. But if we're talking about a eukaryote, then we have to do a little bit of processing. If we're talking about a eukaryote, if this is a prokaryote right over here, this would be our mRNA. If this is s eukaryote, then this is our premRNA, which now has to be processed. And you might say, "Well how is that going to be processed?" Well, there's a couple of things that are going to be done. Some things are going to be added at the beginning and the end of the mRNA. The five prime cap, this is a modified guanine, modified guanine right over here, which is going to help in the translation process as the ribosomes attach onto it. And then you have this polyA tail, and it's called a polyA tail because it has a bunch of adenines at the end, right over here. These not only help in the translation process, it helps make sure that the information is more robust, that the ends of the mRNA don't in some way become, or makes it less likely that they're going to become damaged. Now the other thing that needs to be processed, and this is one of those fascinating things in evolutionary biology, is that we will have in this mRNA sequence, you're going to have parts of the sequence, which we currently consider to be nonsense sequence. Nonsense sequences, and we call them introns. I'm gonna put it in quotes because in general in evolution it's seldom that things have absolutely no purpose, but these are not coding for the protein that is going to be coded by our initial gene. And so, these are actually processed out, they are spliced out. I'm not going to go into the details of the actors that cause the splicing, but as part of this eukaryotic processing, you add the cap, you add the tail, and then you splice out the introns, and once you've spliced out the introns all you have left are the exons. So you have that. It's going to be connected to that. It's going to be connected to that. And so this is what you have resulted. This is in a eukaryote, you will have this mature mRNA. And that's what we saw right over here that can then, let me underline that in a color you can see, right over here, which then migrates out of the nucleus to a ribosome where it can be translated. |
40 | Biomolecules | DNA | Speed and precision of DNA replication | In the earlier video on DNA replication, we go into some detail about leading strands and lagging strands and all of the different actors, all of these different enzymatic actors. But I left out what is probably the most mindboggling aspect of all of this, and that's the speed and the precision with which this is actually happening. As we talked about in that video, it feels pretty complex. You have this topoisomerase that's unwinding things, the helicase is unzipping it. Then you have the polymerase that can only go from the five prime to three prime direction, and needs a little primer to get started, but then it starts adding the, it starts adding the nucleotides. On the lagging strand, you have to have the R, you get the RNA primer, but then it's going from, once again, from five prime to three prime, so you have these Okazaki fragments. And all of this craziness that's happening, and remember, these things don't have brains. These aren't computers. They don't know exactly where to go. It's all because of the chemistry. They're all bumping into each other and reacting in just the right way to make this incredible thing happen. Now what I'm about to tell you is really going to boggle your mind. Because this is happening incredibly fast. DNA polymerase has been clocked, at least in E. coli, has clocked at approaching 1,000 base pairs per second. I think the number that I saw was 700something base pairs per second. So polymerase, let me write this down. This is worth writing down, because it's mindboggling. Gives you a sense of just how amazing what the machinery in your cells are. So it's been as high as, and it can change. It can speed up and slow down, and that's actually been observed. But polymerase as fast as, as fast as 700plus base pairs per, per second. So if this, on this diagram, it, man, it's just zipping, it's just zipping along, at least from our perceptual frame of reference. A second seems like a very short amount of time to us, but on a molecular scale, these things are just bouncing around and just getting this stuff done. Now the second thing that you might be wondering, okay, this is happening fast, but surely it has a lot of errors. Well, the first thing you might say, well, if it had a lot of errors, that would really not be good for biology, because you always have, you have DNA replicating all throughout our lives. And at some point you just have so many errors that the cells wouldn't function any more. And so lucky for us that this is actually a fairly precise process. Even in the first pass of the polymerase, you have one mistake, you have one mistake, let me write this down, 'cause it's amazing. One mistake for every, for every approximately 10 to the seventh. So this is 10 million, 10 million in nucleotides. Nucleotides. And that might seem pretty accurate, but you gotta remember, we have billions of nucleotides in our DNA. So this would still introduce a lot of errors. But then there's proofreading that goes back and makes sure that those errors don't stick around. And so once all the proofreading takes place, it actually becomes one mistake, one mistake for every approximately 10 to the ninth nucleotides. So approximately, you can, it would do this at an incredibly fast pace, as fast as 700plus approaching 1,000 base pairs per second. And you have one error every billion nucleotides, especially after you go through these proofreading steps. And so it's incredibly fast, and it's incredibly precise. So hopefully that gives you a better appreciation for just the magic that's literally, I would look at your hand, or just think about, this is happening in all of the cells or most of the cells of your body as we speak. |
41 | Biomolecules | DNA | Translation (mRNA to protein) | So we already know that chromosomes are made up of really long strands of DNA all wound up into themselves, so something like, well I'm just kind of drawing it as a random long strand of DNA all wound up in itself. And on that strand, you have sequences which we call genes, so that might be one gene right over there, this might be another gene, that might be a gene right over there. And each of those genes can code for specific polypeptides or specific proteins. And the key question is, is how do you go from the information encoded in these genes, encoded as sequences of DNA, how do you go from that? How do you go from the gene, which is encoded in DNA, how do you go from that to protein? Which is made up of polypeptides, which are made up of amino acids. And this is often called the central dogma of biology, but we already saw in the video of transcription, that the first step is to go from the gene to messenger RNA, that the RNA, the messenger RNA, you can use as a transcript, we have rewritten the information now as RNA. And then the next step which we're gonna dive into in this video is going from that message RNA to protein, and this process is called translation, because we're literally translating that information into a polypeptide sequence. And you can see a little bit visually here, this is all review, we covered a lot of this in the video on transcription and the overview video on transcription and translation, is if you look at a eukaryotic cell and the bacteria in a prokaryotic cell, it's analogous, you just don't have the nuclear membrane, and you're not gonna do the processing step that I'm gonna talk about in a little bit and we went in detail on the video on transcription. But you start with the DNA, you have your RNA polymerase as the main actor that's able to transcribe the RNA from that. If we're talking about a eukaryotic cell, what you end up with we wouldn't call mRNA we would call premRNA, premRNA, which then needs to be processed, the introns need to be taken out, we add a cap and a tail here, and if we're talking about a eukaryotic cell, we then formally call that mRNA, and then it can travel, and this is where we get into the translation step. It can travel to a ribosome, which is where it will be translated into a polypeptide sequence. And you see the analogous thing happening here in this bacterial, or this prokaryotic cell right over here, except you don't see the nuclear membrane, because it's prokaryotic, and you don't see that processing step, so you could just consider this straight, this is mRNA right over there. So the questions are well how does this thing happen? And what even is a ribosome? So let's zoom in a little bit on a ribosome right over here, and there's a couple of interesting actors. One, as you can imagine, is the ribosome itself, and it is made up of proteins, proteins plus ribosomal RNA. So in the video on transcription, we're already familiar with messenger RNA and we often view RNA like DNA as primarily encoding information, it's acting as a transcript for a gene, but it doesn't have to only encode information. It can also, so it's proteins plus, it's not a 'T' there, this is a plus. It can also provide a functional structural role, which it does in ribosomal RNA. And this big, you know, this looks like a an oversized hamburger bun or something right over here, this is super oversimplification of what a ribosome looks like and I encourage you to do a web search for image searches for ribosomes, and then you can get more appreciation of how how beautiful these structures are, and how intricate they actually are. So this is the site, and you can broadly think of the ribosome as having this, you know, this is the top bun, and the bottom bun. And it's going to travel along the mRNA from the five prime end, to the three prime end, reading it, and taking that information, and turning it into a sequence of amino acids. So how does that actually happen? Well, each, each of these three, every three nucleotides, every three nucleotides there, we call that a codon, so that's a codon, this is, let me do this in a color that is visible on both white and black. So these next three nucleotides is a codon, this is a codon, this is a codon, and what's actually the information is actually encoded in the nitrogenous bases. So this first codon right over here, we see it's AUG, so the nitrogenous bases are adenine, uracil and guanine. And this has, this codon, it codes for the amino acid methionine, but this is also, this is a good one to know, AUG, let me write it over here. AUG is know as the start codon. Start codon. This is where the ribosome will initially attach to start translating that messenger RNA, and so we, the way this drawing is, that we are just starting to translate this messenger RNA. So how does that actually happen? How do we get from these three letter sequences to specific amino acids? Well let's think about it, how many, how many possible three letter sequences are there? Well, there are, there are four possible nitrogenous bases there, so there's four possible, so if you, if you have a codon, and it has three places, there's four possible things that could be in the first place. There's four possible things that could be in the second place, and there's four possible things that could be in the third place. So there are 64 possible permutations. 4 times 4 times 4. Permutations, so you can think of it, there's 64 different codons, different ways of arranging the A, the U and the G. And that's good, because there are many amino acids, and this is actually overkill, because there's actually 22 standard amino acids, 22 standard, amino, amino acids, and 21 that are typically found in eukaryotic cells. So we have more than enough, more than enough permutations to cover the different amino acids. And it's not hard to find tables that will actually show us what the different sequences, what they actually code for. So you can see here, you can take the first letter, the second letter and the third letter, figure, look at the different sequences, and you can say, okay, look at that. AUG, adenine, uracil, guanine. That codes for methanine. Right over here. You could do that with any of them, you could say cytosine, uracil, uracil, that codes for leucine. And you can see that it's not just one amino acid per codon, but here you have four codons all code for, all code for leucine. And so it turns out that 61 of the codons, let me write this down. So 61 of the codons, of the possible 64, code for amino acids, amino acids, and three play a role that essentially tells the the ribosome to stop, three codons, three codons are stop codons, and you can see them right over here. UAA, UAG, UGA, that's how the ribosome knows to stop translating. So AUG, that's a start codon, and it codes for methionine. So that lets you know that these polypeptide chains are going to start with methionine, and then these characters tell it where to stop. But how do, how does the amino acid actually get, how do they all get tied up together to form this polypeptide? And how do they get matched up, how do they actually get matched up with the appropriate codon? And that's where we have another RNA based actor, and this is tRNA. So tRNA, the t stands for transfer, transfer RNA. There's a bunch of different tRNAs that each combined to specific amino acids, and on parts of those tRNA, they have what are called anticodons. That pair with the appropriate codon. So this tRNA, and that's not what it looks like, I'll show you in a second what it looks like. That's a tRNA molecule, tRNA, at one end of the molecule, it's binding to the appropriate amino acid, methionine, right over here. And then at the other end of the molecule, though that's in the middle of the tRNA actual chain, you have your anticodon. And your anticodon matches up to the appropriate codon. And so this is how they bump into each other the right way and the ribosomes going to facilitate it, that the AUG is going to be associated with the methionine. And if we look at what tRNA actually looks like, and this is still just a visualization. So this is a strand of tRNA, you get a sense of, okay, it's a sequence of RNA right over here, this it's, I guess you could say, you could think of it, it's two dimensional structure. But then it wraps around itself to form this fairly complex molecule. And the anticodon, which is right here, it's kind of in the middle of the sequence, it forms the basis for this end of the molecule, that's the part that's gonna pair with the codon on the mRNA, and then at the other end of the molecule, at the other end of the molecule is where you actually bind to the appropriate amino acid. So I know what you're thinking, alright, I see that the ribosome, it knows where to start, it starts at the start codon. I see how the appropriate tRNA can bring the appropriate amino acid, but how does the chain actually form? And you can view this in three steps, and associated with those three steps are three sites on the ribosome. And the three sites, we call this the Asite, you're not gonna be able to see it if I write it in black. A, or yellow, alright, let me write it in blue. So that is the Asite. This is the Psite, and this is the Esite. And I'll talk in a second why we call them A, P and E. So the Asite is where the appropriate tRNA initially bounds, the tRNA that's bound to an amino acid. And so you can see, we're starting the translation process, the next thing that's going to happen is another tRNA, the one that is, that matches, that has an anticodon that matches the UAU, that's going to bond over here on the Asite, and it's bringing the appropriate amino acid with it, it's bringing the tyrosine with it. So why is that called the Asite? Well A stands for aminoacyl. An easy way to remember it it's the tRNA, it's the place where the tRNA that's bound to the amino acid, just one amino acid is going to bind on the ribosome. And so once that happens, once this character comes here, let me draw that. Once this character comes right over here, it's gonna be AUA, and it's bound to the tyrosine. Well then you could have a peptide bond form between the two amino acids, and the ribosome, and the ribosome itself can move to the right. So this, this tRNA will then be in the Esite. This tRNA will then be in the Psite, and then the Asite will be open for another amino acid carrying tRNA. So what this, what do the P and E sites stand for? Well you can see a little bit more clearly right over here. So the Psite is where you have the polypeptide chain actually forming, and, so the Psite is often, well, one way to remember it is is that's where you have the polypeptide chain, and now you have a new, you have a new Asite where you can bring in a new amino acid. And then the ribosome is going to shift, once this is bound, the ribosome, the peptide bond forms, and then the ribosome can shift to the right, when the ribosome shifts to the right, we're gonna be in this position, where the thing that was here, that was in the Asite, now the polypeptide is attached to it, it is now going to be in the Psite, and the thing that was in the Psite is now going to be in the Esite. It is now ready to exit, and that's why it's called the Esite. Because that's the site from which you exit. And so this is going to keep happening until we get to one of the stop codons. And when you get to one of the stop codons, then the appropriate polypeptide is going to be released, and we will have created this thing that could either be a protein, or part of a protein, so this is very exciting, because this is happening in your cells as we speak. This is, and in fact if you think about things like antibiotics, the way that they work are, is that, or the way that antibiotics work is that ribosomes and prokaryotes are different enough than ribosomes in plants and animals or in eukaryotes, that we can find molecules that hurt the function of ribosomes in prokaryotes, but don't do it to eukaryots. And so if you have bacteria in your blood stream, and if you take the appropriate antibiotic, it could disrupt this translation process in the bacteria, but not in your cells that you want to keep. |
42 | Biomolecules | DNA | Differences in translation between prokaryotes and eukaryotes | Let's talk about some of the differences between how translation happens in prokaryotic cells and how it happens in eukaryotic cells. And I want to focus mainly on the mRNA just before it's ready to be translated. So let's start with our prokaryotic mRNA and let's look at our five prime side first. So we have this yellow part right here, and that's the noncoding region. And it's called the noncoding region because the ribosome is not actually going to read that part. So that particular sequence of amino acid is not that important. And then after the noncoding region we have the ShineDalgarno sequence. And the ShineDelgarno sequence is the site that the ribosome's going to recognize and bind to. So let's just throw a ribosome right over here. This is where the prokaryotic ribosome is going to bind. And then after the ShineDelgarno sequence, we have another noncoding region. Just gonna abbreviate it NCR. And then we have our start codon, which is typically AUG, so that tells us to start. And so the ribosome's going to start translating, it's going to read this entire section, put together the corresponding polypeptide chain, until it hits the stop codon, which tells it to stop translating. And then we have another noncoding region. Let's look at our eukaryotic mRNA. And so it's pretty similar, but you can see there are some differences. So we'll start with our five prime side first. So you see this red nucleotide right over here. That's the five prime cap. And the five prime cap is simply a guanine nucleotide. So I'm gonna draw a G inside, Guanine, and it's going to have a methyl group somewhere on the molecule. So I'm gonna draw a methyl group. And the bond between this guanine and the nucleotide right near it is a bond that's different than the bond that you'd typically find between two nucleotides. And so that's really all the five prime cap is. And the five prime cap is actually the ribosomal binding site in eukaryotes. So that means that in eukaryotes, the ribosome's going to recognize this particular part and bind to it. So after the five prime cap, we have this other noncoding region which the ribosome's not going to translate. And then the ribosome is going to hit the start codon again. AUG tells it to start, and it's gonna start translating, so it's going to translate this entire section until it hits the stop codon. And then we have another noncoding region. And then we hit something that looks different than what we've seen in the prokaryotic mRNA, so this section with blue nucleotides, and that's called the polyA tail. And the polyA tail is a bunch of nucleotides that are all A's, or adenines, so I'm gonna draw A's inside all of these nucleotides. And the polyA tail is actually pretty long, so it's typically anywhere between 100 and 250 nucleotides long. So that's pretty long. So I didn't exactly draw it to scale. And the purpose of both the five prime cap, and the polyA tail is to prevent this mRNA from being degraded by enzymes. So it acts as kind of a signal that does not allow enzymes to break it down or degrade it. And so you might be wondering, well, what about prokaryotic mRNA? How come they don't have anything similar to prevent them from being degraded. And the brief answer to that question is that in prokaryotic cells, transcription, that's an R, and translation, both happen in the same place. So prokaryotic cells don't exactly have a nucleus. They have this cytosol and transcription and translation are happening in the same place. And not only are they happening in the same place, but they can actually be happening at the same time. So you can have a piece of mRNA that's being formed, and while it's being formed, a ribosome will attach to it and being to translate it. But, in eukaryotic cells, things are a little bit different. So transcription... happens in the nucleus, and translation happens in the cytoplasm where there are ribosomes. And so the mRNA, after it's made, has to travel, from the nucleus to the cytoplasm to where the ribosomes are. And so because it's traveling this relatively large distance, it's going to encounter a lot of different things, including enzymes that might break it down. And so it needs this extra protection to prevent it from being damaged in any way. There's one more difference I want to talk about in how translation happens in prokaryotes and eukaryotes and that is what the first amino acid in the polypeptide chain will be. So in prokaryotic cells, the first amino acid in the chain is always formylmethionine. And formylmethionine is simply the amino acid methionine, but with a formyl group attached. And in case you don't remember what a formyl group looks like, it looks like that. In eukaryotic cells, the first amino acid in all the polypeptide chains is simply methionine. And it's interesting to note that formylmethionine actually acts as an alarm system in the human body. So if you had some bacterial cells in your body that were damaged in any way, there would be these formylmethionines floating around, and that tells your body that there are bacteria around, and it's going to trigger an immune response. |
43 | Biomolecules | DNA | DNA repair 1 | Let's take a look at a segment of DNA that's in the process of being replicated. I want to focus in particular on the enzyme that replicates DNA. That enzyme is DNA polymerase. Actually there are a few different types of DNA polymerases, and the one that we're looking at right now is DNA polymerase III. DNA polymerase III synthesizes new DNA, and it also has the ability to proofread, or kind of check the DNA it's putting together and make sure there are no mistakes in it. But before we get into that, let's just orient ourselves and quickly summarize the diagram that we're looking at. This enzyme over here is DNA helicase. That's the enzyme that unwinds the doublestranded DNA, so that DNA polymerase can then come in and started replicating. Right over here you can see I drew the backbone in a different color. That's the RNA primer. Let's just label the DNA strand that's being synthesized, it's synthesized from 5 prime to 3 prime. Actually, the bottom strand of DNA is synthesized in the same time as the top strand, but I just left that out of the drawing to keep things simple. Let's say that the yellow bases represent the nitrogen base thymine. Let's say that the orange bases represent cytosine. The green ones represent adenine. And the blue ones represent guanine. Thymine and cytosine are the pyramidines. They are composed of a single ring structure, so they're made up of one ring that has six sides to it. Then adenine and guanine are the purines. They are a doubleringed structure. They're composed of one ring with six sides to it, and then that ring is attached to another ring that has five sides to it. Actually, these structures are a little bit more complex. There are other atoms in it, and there are some double bonds, but we're just going to keep things simple for now and leave it at that. Let's get back to our DNA that's being replicated. Right over here I left a space. I didn't put the nucleotide in. Let's say that by accident, instead of it being paired up with the proper base, which is adenine, it accidentally gets paired up with a guanine. That's a mistake. DNA polymerase III actually has the ability to sense if it made a mistake, and if it does realize that it's going to go backwards. It's going to actually remove the incorrect base and replace it with the correct base. So let's do that. It's going to remove the incorrect base and replace it with the correct one. Of course, remember the nitrogen base is attached to the sugar backbone. This activity that I just described to you is called exonuclease activity. Nuclease, that tells us that means the ability to remove a nucleotide. Exo, just going to underline that. Exo tells us that it can remove a nucleotide, but only from the end of a DNA strand. It was able to remove the nucleotide because it was at the end of a strand. This is in contrast to endonuclease activity. An endonuclease can actually remove a nucleotide from the middle of a DNA strand. So it would be able to remove a nucleotide from right over here, for example. Just keep that in mind because we're going to come across some endonucleases as well. Anyway, back to our exonuclease activity. If we want to be more specific, the exonuclease activity of DNA polymerase III is actually 3 prime to 5 prime exonuclease activity. The reason it's called 3 prime to 5 prime exonuclease is because when DNA polymerase 3 makes that correction it has to move backwards in the 3 prime to 5 prime direction in order to do that. There's another enzyme, DNA polymerase I. I'm just going to abbreviate polymerase with POL. DNA polymerase I also has exonuclease activity. DNA polymerase I is actually the enzyme that will remove the RNA primer at the end of replication. Just as a side fact, the exonuclease activity of DNA polymerase I is actually in the 5 prime to 3 prime direction. If you want, you can just keep that in mind. DNA polymerase III and DNA polymerase I are both able to repair or fix mistakes that happen during DNA replication. Just to give you some perspective as to how often this occurs with and without repairs, normally we'll have a mistake happening in replication between 1 in 100,000 bases to 1 in 1 million bases. That's normally the amount of mistakes that would occur. But, with the repair mechanisms of DNA polymerase III and DNA polymerase I, this is reduced to a mistake that happens once in about 100 million bases. They are very, very effective at lowering the error rate in DNA replication. The next question I want to ask is what if this mistake over here was somehow not corrected during replication? Maybe there was something wrong with one of the enzymes, something happened and that mistake was actually sustained. Let's take a look at that. Here is a piece of DNA with our mistake incorporated into it. Before we discuss if this mistake can be corrected or not, let's see what happens if this mistake is not corrected. Right here we have our original DNA. We're replicating it. Let's just say that this strand over here is the same as that strand. Let's say that the bottom strand in our original DNA is the same as this strand. Let's look first at the newly replicated DNA on the left. We have right over here a thymine base. Assuming DNA was replicated properly, it's going to have an adenine complementary to it. Now let's take a look at the DNA on the right. On the bottom we had a guanine, and it's going to be paired up, hopefully, with the correct base, which is a cytosine. Now, let's just quickly look back at our original DNA. We were supposed to have a thymine with it's complementary adenine, and actually, that's exactly what we got over here. Just going to circle it. So this DNA is actually in the correct sequence. But look at the DNA over here on the right. This is not correct. This is a mutation. This is an example of how mutations can occur if the DNA repair mechanisms are not working properly. Let's go back to our original question. Can we fix the original mistake so that this mutation does not occur. The answer to that question is yes. Fortunately, our cells have what's called the mismatch repair mechanism. The mismatch repair mechanism is composed of a number of proteins. The first thing these proteins are going to do is they're going to recognize if there's a problem. The reason that they're able to recognize the problem, is that when you have a mismatch in DNA it tends to distort the sugar backbone a little bit. They are going to mark the area with a cut. They are going to cut the incorrect base or mark it with a cut. The next thing that's going to happen is an exonuclease is going to remove the incorrect nucleotide. So we're going to remove the incorrect nucleotide. The next step is one of the DNA polymerases is going to insert the correct nucleotide. So we're going to pair our thymine up with adenine. The last step is a DNA ligase is going to connect the new nucleotide to the nucleotides on its sides, and also to its complementary nucleotide on the other strand. I'm actually going to just correct that distorted sugar backbone. Here's our repaired DNA. Just to clarify, the mismatch repair mechanism that we're talking about here happens after replication. The repairs done by DNA polymerase III and DNA polymerase I that we discussed before, that happens during replication or at the end of replication. One thing you might be wondering is how does the mismatch repair mechanism know to distinguish between the original parental strand and the newly synthesized strand that has the mistake on it? In other words, how does it know which base over here is correct, in our case that's the thymine, and which one is incorrect, in our case, well, it was a guanine. We know the answer to that question in bacteria. In bacteria, the parental strand will have adenines that are methylated. I'm just going to draw some methyl groups on all the adenines. That allows the mismatch repair mechanism to kind of recognize and distinguish between the original strand that has the correct base on it, and the new strand that has the incorrect base on it. But we're not quite sure how the mismatch repair mechanism in eukaryotic cells and in other prokaryotic cells knows to distinguish between the strand that has the correct nucleotide and the strand that has the incorrect nucleotide. |
44 | Biomolecules | DNA | DNA repair 2 | There are certain things that can cause damage to the structure of DNA, and one example of that is UV rays. And so, before we get into the damage that UV rays cause, let's just focus for a second on the key that I drew here on the left, and it's just to help us remember which colors represent which nitrogen bases. So, yellow represents the nitrogen base thymine. The orange bases represent cytosine. The green bases represent adenine, and the blue bases represent guanine. And so back to our DNA that's being damaged. If you look over here, there are two thymine bases that are kind of stuck together, and that's called a pyrimidine dimer. So, a dimer is simply when you have two molecules that are identical that are stuck to each other, and pyrimidine tells us that it can be two thymines that are stuck together, or it can be two cytosines that are stuck together. And UV rays cause the formation of pyrimidine dimers. And so you can see that the pyrimidine dimer actually is causing the sugar phosphate backbone to protrude, or kind of stick outwards, and not just that, but because the backbone is sticking outwards, the bond between this cytosine and this guanine snapped. So, you can see that there's some structural damage that happened to the DNA. And so, what are some factors that can cause damage to DNA? And, I want to just make a clear distinction between a mutation and DNA damage. So it's not the same thing. A mutation is when you have a change in the sequence of DNA. So, for example, if we had a piece of DNA that read ATCG, and then something happened and it read AACG. So this A is in the wrong place. That is a mutation. But when we talk about DNA damage, we're talking about damage to the structure of DNA, but the nucleotides are actually in the correct order. And so, DNA damage can be caused by Endogenous, or internal factors, and that means factors that originate within us, within our own cells. So, for example, there are certain byproducts of metabolism that can cause DNA damage, or DNA damage can be caused by Exogenous, or external factors, and those are factors that originate outside of us, or outside of the organism that we're discussing. So let's start with Exogenous factors first. So, we spoke about one of them, UV rays, and there are a lot of Exogenous factors that cause DNA damage, but we're just gonna list a few. Gamma rays can cause DNA damage. Xrays, and so that's why it's not healthy to be exposed to a lot of these rays. And, now let's talk about some Endogenous factors. So, reactive oxygen species is an example of an internal factor that can cause DNA damage. In a reactive oxygen species are molecules that contain oxygen and they're highly, highly reactive. So, there are a lot of different kinds of reactive oxygen species, but we're just gonna give two examples. So, for example, a super oxide anion, which is O2 with a negative charge. So let's just draw that. It's two oxygen atoms that are bound together, but there's one extra electron. And I'm actually gonna draw the extra electron in a different shade of purple, and so this whole molecule has a negative charge. So that's a reactive oxygen species. Another example would be peroxides. So peroxides are molecules that have two oxygens, and on either end, there's another atom. So that R can represent different types of atoms. So this is the general way that a peroxide looks. You might've heard of hydrogen peroxide. So this is hydrogen peroxide. And so where are these reactive oxygen species in our cells coming from? So actually, reactive oxygen species are a normal byproduct of the electron transport chain in the mitochondria. So there are a lot of reactive oxygen species all over our cells, but, fortunately, we have many enzymes that help protect against the damaging effect of reactive oxygen species. And, you may have heard of the term antioxidant, and so, an antioxidant is a molecule that also helps protect us against the damaging effect of reactive oxygen species. You may have heard that certain foods are really healthy because they have a lot of antioxidants, and that's true. So, vitamin C, for example, is an antioxidant. Vitamin E, and there are many, many different types of antioxidants, but we're just gonna give these two as an example. And so now that we've discussed some of the sources of DNA damage, let's go back to our damaged DNA and see if there's a way to fix this. So our cells can get rid of the pyrimidine dimers in a process called nucleotide excision repair. And so, the first step in nucleotide excision repair, is an enzyme, an endonuclease, is going to remove the pyrimidine dimers and any other nucleotides that are kind of not the way they're supposed to be. And so I just want to pause for a second and analyze that word. So, nuclease tells us that it's an enzyme that's able to cut out nucleotides, and that prefix endo tells us that it's able to cut out nucleotides from within a DNA molecule. That's in contrast to an exonuclease that can only take out nucleotides that are at the beginning or end of a DNA molecule. But anyway, the endonuclease is going to cut out the dimer and any other nucleotides that are not properly arranged. So let's just cut out all these nucleotides. The next step is a DNA polymerase, I'm just going to abbreviate that pol, is going to come and bring the nucleotides that belong there. And then the last step is DNA ligase is going to make sure that those new nucleotides are attached properly to the nucleotides on either side and also the nucleotide that's complementary on the other strand. And so, that was a mouthful, but let's actually just draw all of that. So let's get rid of our backbone that's kind of protruding and just not right. So let's redraw our backbone. Something like that, or actually, draw it a little bit closer, and then, DNA plumerase brings the correct nucleotides, but remember, it's the ligase that actually connects the nucleotides properly. And so here's our corrected DNA. But, what happens if, for some reason, the nucleotide excision repair is not working properly, and this repair mechanism is only one example, there are many different types of DNA damage that can occur, and many different types of repair mechanisms. What happens if, for some reason, one of these, or a couple of these, are not working properly? Then, we get a cell that has a lot of damaged DNA. And there are three things that can happen to a cell like this. The first is it might go into this dormant state, where it just ages and does not divide any more. That's called senescence. The second thing that might happen to it is what's called programmed cell death, or apoptosis, and that basically means that the cell's going to commit suicide and die. And the third thing that might happen is the cell might start to divide uncontrollably. So I'm gonna write unregulated cell division. And this can cause cancer. And so actually, the skin cancer melanoma is an example of this. Melanoma, that's an n, melanoma happens when the nucleotide excision repair mechanism that we just discussed is not working properly, and so you have this accumulation of pyrimidine dimers that damages the DNA very much, and then the cell starts to divide uncontrollably. |
45 | Biomolecules | DNA | Semi conservative replication | Let's take a piece of DNA. And let's just unwind it into its two strands. And just to help us to remember that DNA is a very, very long molecule, I'm gonna put arrows here on our two strands of DNA. And the question I want to ask you is, "If we were to replicate this DNA, "what would the end result look like?" So, I'm kind of skipping over the entire process of how the DNA is replicated and focusing just on the product. And so we have three choices. The first is conservative replication. And in conservative replication we have our old pair of DNA and then we synthesize a completely new pair of DNA. So you can see the old pair, that looks just the same as what we had before, in yellow. And then we have a completely new pair which is represented in blue. Our next choice is dispersive replication. And in dispersive replication, we're gonna end up with two pairs of DNA. And in each one of those pairs we have some old DNA and new DNA dispersed within that double strand of DNA. So you can see there's yellow and blue mixed up together. And it wouldn't necessarily have to be in the ratio that I drew it in. I drew it in this kind of neat ratio where the yellow and blues are the same size. But perhaps the yellows would be a little bit bigger and maybe some of the blue parts smaller or vice versa. And the third option we have is semiconservative replication. And in semiconservative replication, each pair has one old strand, that you see in yellow, of course, and one new strand, that's in blue. And this question was answered by two scientists. One by the name of Meselson and one by the name of Stahl. And they conducted a famous experiment which was named after them. So the Meselson Stahl experiment. And in this experiment they proved that DNA replication is semiconservative. So, this is how DNA is replicated. |
46 | Biomolecules | DNA | Protein modifications | After a polypeptide chain is formed, it's going to be folded into its secondary and tertiary structure into a very specific 3D conformation or shape. And at this point, we can start calling it a protein. But, this protein may not be ready to carry out its function just yet. There might be some additional protein modifications that need to be made to this protein before it can be functional. And those are called protein modifications. There are two different types of protein modifications. The first type is cotranslational modification. And that means that these are modifications or changes that happen to the protein, or actually to the polypeptide while it's being translated. Let's say we have the ribosome right here and we have a polypeptide that's being formed. So these changes are going to happen while the polypeptide is being formed. An example of a cotranslational modification is acetylation. And what happens during acetylation is, the first amino acid in the polypeptide, which is usually methionine, is going to be removed. And in its place we put an acetyl group. Let's just draw an acetyl group. And acetylation happens to 8090% of eukaryotic proteins. But the significance of this modification is not known very well, we're actually trying to figure out what the purpose of this modification is. The other type of protein modification that happens is posttranslational modification. And actually, most protein modifications fall into this category, and the examples we're going to discuss in this video are all posttranslational modifications. And those modifications happen after translation. Many posttranslational modifications happen in the endoplasmic reticulum and the Golgi apparatus, but not all of them. Let's go through some examples. So the first posttranslational modification I want to talk about is glycosylation. Glycosylation, you can look at the word. The prefix "glyco" tells us that it has something to do with a carbohydrate. And so glycosylation is the adding of a carbohydrate to a protein. And most of the proteins in this video are all in green. And glycosylation usually happens to proteins that end up being embedded in the cell membrane. So you can see here we have a cell membrane and this protein embedded in it, and then there are these carbohydrate groups attached. So here's a carbohydrate group attached, and here's another one. Glycosylation helps to identify different types of cells. And one very common example of where we use glycosylation is in the A, B, O blood groups. Let's take four different red blood cells, and let's just say that each one of these red blood cells belongs to a different person. Red blood cells have these proteins embedded in their surface. And these proteins are going to have, many times, different carbohydrate groups attached to them. So let's say that this person right here has this particular carbohydrate group attached to it. That makes him blood type A. Let's say that this person has a different type of carbohydrate attached to the protein. Let's say it looks something like that. That makes them blood type B. Let's say that this person has both of those carbohydrates attached to his red blood cells. That would make him blood type AB. And let's say that this last person does not have any carbohydrates of this category attached to the proteins on his red blood cells, and so that makes him blood type O. So here's a very common example of how glycosylation is used in the identification of different types of cells. Let's go on to a different type of posttranslational modification that's pretty similar, and that is lipidation. Lipidation is when we add a lipid to a protein, also a protein that's going to be attached to the cell membrane. And this lipid we're looking at is actually an example of a GPI anchor. And GPI anchors are lipids that help to attach or tether proteins to the cell membrane. And just to give you an idea of maybe why this would be necessary. To quickly review the structure of the cell membrane. We have these hydrophilic heads. That means that they are polar. And then we have, inside, these hydrophobic tails, and that means that they are nonpolar. And so the protein has both polar and nonpolar parts on it, and maybe it just doesn't attach well to the hydrophilic portion of the cell membrane. So this GPI anchor, a lipid, kind of plunges into the lipid or hydrophobic part of the cell membrane. And we know that substances that are similar, like substances that are both hydrophobic, attach very well to each other. So this lipid, which is hydrophobic, attaches very well to the inner part of the cell membrane that is also hydrophobic. And so that's how it helps to attach the protein to the cell membrane. Both glycosylation and lipidation usually do occur in the endoplasmic reticulum, or in the Golgi apparatus. Let's move on to some protein modifications that have more to do with the activity or the function of the enzyme and less with the structure. So one very, very common protein modification I want to discuss is phosphorylation. Phosphorylation is basically the adding of a phosphate group to a protein or to an enzyme. Phosphorylation comes along with dephosphorylation, is when you remove a phosphate group from the enzyme or the protein. I'm just going to make a little bit of room over here. And so, what you're looking at is this schematic diagram of the sodiumpotassium pump that's found in basically every animal cell. And another name for the sodiumpotassium pump, which is the enzyme that you're looking at, is the Na+ /K+ ATPase. And so, this enzyme or protein, the sodiumpotassium pump, is responsible for maintaining the proper osmolarity of sodium ions and potassium ions in and out of the cell. And so, let's see how phosphorylation regulates this protein. And again, the proteins that you're looking at, they really all represent one protein. It's just, we're going through the motions of the changes that happen to this one protein. It's not like we're looking at six different proteins in the membrane. So here's our first step. And when you look at this enzyme, you can see that there are three receptor sites for a particular ion, this is the sodium ion. Represented by these dark blue circles. And then there are two receptor sites that look kind of more squarish, and those are receptor sites for the potassium ions, and those are represented by these light blue squares. So those are the potassium ions. And so, back to the first step, and what's happening here. The sodium ions will attach to the receptor sites on the enzyme. And just to clarify, this is the intracellular space, so this is basically the cytoplasm. And so these sodium ions are coming from the cytoplasm. And then out here is the extracellular space, the outside of the cell. Back to our first step. So the sodium ions attach to the receptor site. When the receptor sites are full, it's going to cause something to happen. It's going to cause an ATP molecule, adenosine triphosphate, to break down into ADP, adenosine diphosphate, plus phosphate. And then this phosphate will attach itself to the protein, and that is phosphorylation. And so when this protein gets phosphorylated, when the phosphate group is attached to it, it causes there to be some sort of change in the conformation of the protein. And that change in conformation causes the protein to turn itself around by 180 degrees and face the outside of the cell. That's what you're looking at right over here. So our protein is still phosphorylated. I know I drew it in a different place, but just keep in mind that there's still a phosphate group attached to that protein. And then the protein releases the sodium ions into the extracellular space. So the next step is number four. And again, we are still phosphorylated, there's still a phosphate group attached to our protein. And so in the next step, the potassium ions on the outside of the cells will attach themselves to the protein on the receptor sites. Take note, there are three receptor sites for sodium but only two for potassium. When the potassium receptor sites are full, it's going to cause a different change to happen. This phosphate group is going to be removed. And that is called dephosphorylation. That phosphate group ends up in the inside of the cell, and it gets recycled in some other way. And when this protein is dephosphorylated, when that protein is removed, it's going to cause a different change in the conformation of the protein. And that change in conformation causes the protein now to turn around again by 180 degrees and it faces the inside of the cell. So that's our fifth step, you can see the protein is facing the inside of the cell. And in the last step, the potassium ions are released into the inside part of the cell. And then we're back to our first step. And so you can see that the phosphorylation and dephosphorylation basically regulates the activity of this protein. And the end result that we're trying to get to is that we want there to be, on the outside of the cell, a rather high concentration of sodium, and a rather low concentration of potassium. And on the inside of the cell we want there to be a rather low concentration of sodium, and a relatively high concentration of potassium. That's accomplished by the fact that for every three sodiums that are pumped out, two potassiums are pumped in. And so here's an example, a very common example of how phosphorylation regulates a protein. And this doesn't happen just in the sodiumpotassium pump, it happens in many, many enzymes and proteins in our bodies and in cells. Let's move on to two other protein modifications that also have to do with regulating an enzyme or something similar to that. So the next protein modification I want to talk about is methylation. And in particular, the methylation of certain proteins called histones. And those are these green circles that you're looking at. Histones are these proteins around which DNA wraps itself. So they're found in the chromosomes, and they help to package DNA in a very tight and organized manner. And sometimes histones are methylated, so let's put some methyl groups on our histones. Methylating and demethylating histones helps to turn certain genes on and off. And so here's another example of, a protein modification helps to regulate activity, but in this case we're regulating the activity of genes as opposed to proteins. Another protein modification I want to bring up is proteolysis. And by looking at this word, "proteo" means protein and "lysis" means to break something down, or to cut something. Proteolysis is sometimes to take a protein and activate it, we need to cut it. And in fact, the insulin has to be cut twice before it's activated. So let's cut this protein twice. You may have actually heard of the term zymogen. A zymogen is an inactive form of an enzyme. And sometimes the way to activate a zymogen is by cutting it, and that's proteolysis. There's one more protein modification I want to discuss. And that is, sometimes we add a protein, ubiquitin, to another protein. So that protein I just added is ubiquitin. And this process is called ubiquitination. And what ubiquitination does is, it basically marks this green protein for degradation, or for breakdown. So within a short while of being ubiquinated, this protein is going to be destroyed and the different parts are going to be recycled. Let's just quickly review the posttranslational modifications that we discussed. So we talked about glycosylation and lipidation. Those are two protein modifications that generally happen to proteins that end up being embedded in the cell membrane. So glycosylation was the adding of a carbohydrate group which helps to identify certain cells, and lipidation is when you add a lipid to a protein, and that generally helps to anchor a protein to the cell membrane. Then we talked about phosphorylation, methylation and proteolysis. And these all had to do with activating or deactivating an enzyme or genes. So phosphorylation, we brought the example of the sodiumpotassium pump, which is basically regulated with phosphorylation and dephosphorylation. Then we spoke about methylation, which basically helps to turn on or turn off certain genes. And proteolysis is a way in which many enzymes are activated. And the last posttranslational modification we talked about was ubiquitination. And ubiquitination marks a protein for degradation, and then the various parts are recycled. |
47 | Biomolecules | DNA | Jacob Monod lac operon | So, hopefully by now you're familiar with the central dogma of molecular biology that tells us that DNA makes RNA in a process known as transcription and RNA makes protein in a process known as translation. Well let's take a look at two cells. Let's say that right over here we have an eye cell and let's say that we have a skin cell. And so, what makes the eye cell an eye cell and what makes the skin cell a skin cell? So, they both have the exact same DNA because all the cells in our body or in any organism have the same DNA. And so what makes an eye cell an eye cell and a skin cell a skin cell is which genes are expressed in that cell, so in the eye cell we have the expression of genes that make certain proteins that are unique to an eye cell and in a skin cell, we have genes that are expressed and they make proteins that are unique to a skin cell. And so the question I want to focus on is how does that happen? How do we regulate the expression of genes so that only those proteins that are necessary for the cell get expressed or are made. So let's just frame our question. How is gene expression regulated? So, let's look at the options that we have. Maybe gene expression is regulated at the protein level. What that means is that the DNA in each cell is all transcribed into RNA and then all of the RNA gets translated into proteins. So, according to this model, you'd have in each cell all of the proteins coded for by the entire human genome, if we're talking about humans, but then only those proteins that are necessary for the cell get activated. So, for example, in the eye cell, all the DNA gets transcribed into RNA and they all get transcribed into proteins, but only the specific eye proteins are activated and all the other proteins are inactivated. Maybe that's what happens. Maybe gene expression is regulated at the level of translation. That would mean that all the DNA in the cell is transcribed into RNA, but not all the RNA gets translated into protein. Just the RNA that make proteins of that particular cell would get translated. And the third option we have is maybe gene expression is regulated at the level of transcription. And that would mean that not all the DNA gets transcribed into RNA. Only the DNA that codes for proteins for that specific cell would get transcribed into RNA. So, for example in the eye cell, you'd only have DNA transcribed if that DNA is a gene that codes for a particular eye protein. And the answer to our question is that usually gene expression is regulated at the level of transcription. And if we think about it, this should make sense, because this is really the most effective way for a cell to make use of its resources. So, let's take a look again at our, you know, if gene expression was regulated at the protein level. Again, say we're talking about humans, ourselves. So, that would mean that in each of our cells, all the DNA gets transcribed into RNA and then all of that gets made into protein. So, we would have a tremendous amount of protein in our cells and actually thinking about it, I don't even think there's room in one cell for all of the proteins coded for in the genome. But even if there was, that would be a huge waste of energy. It takes a lot of ATP to put together proteins and so why would we want each cell to make a whole bunch of proteins that they'll never even use. So this is not very efficient. What about translation? If we regulated gene expression through translation? Well, it's more efficient than the protein level, but it's still also not that efficient because that means we have a bunch of RNA that would never even get translated, so making RNA doesn't take as much energy as making protein, but still, that would be a big waste of energy. And so it turns out that regulating gene expression at the transcription level is the most efficient because we're not making any RNA or any protein that we're not going to use. And we actually have a lot more to learn about how gene expression is regulated, but there's a particular model that we understand pretty well thanks to the work of two French scientists by the name of, one of them was Francois Jacob and the other one was Jacques Monod. And they discovered the mechanism of the lac operon. So, we call it the Jacob Monod lac operon. And the lac stands for the word lactose and the lac operon is found in the bacteria e. coli so it's a prokaryotic cell. And the picture that you're looking at is a sketch of the lac operon. It's a section of DNA in e. coli and let's just label the diagram so that we orient ourselves. So, let's say that this is the coding strand. Which means that this is the noncoding, or the template strand. And if you recall, it's actually the noncoding, or template strand, that gets transcribed, and that's the reason that I color coded the noncoding strand with various genes. Each of these colors represent a gene and we'll explain in a minute what they are. Now if I wanted to be more exact maybe I really should've also color coded the top because these two strands are complementary to each other. But I'm not gonna fill that out. Just use your imagination and remember these are complementary and I also just want to point out that I drew this transcription bubble because it's going to be easier for me to show you what's going on in that way, but the default is that these two strands are really stuck together and you usually do not have this bubble forming unless transcription is happening. So, just keep that in mind as we go along. Okay, so what is this lac operon? So, before we talk about the details, the lac operon has a couple of genes that will make enzymes that help e. coli break down lactose. So, let's take a look. So over here we have these three genes. They are called structural genes. It's not important for you to remember that. But, these three genes, this here is the lac z gene. This is the lac y gene, and this is the lac a gene. And so if you recall, the sugar lactose gets broken down into glucose, and galactose. So glucose and galactose are monosaccharides and lactose is a disaccharide. And the lac z, lac y, and lac a genes are all each going to code for an enzyme that helps in the breakdown of lactose, or in the metabolism of lactose. So, let's look at the lac z gene. The lac z gene codes for a protein, beta galactosidase. And beta galactosidase is the enzyme that actually breaks lactose down into glucose and galactose. The lac y gene codes for the enzyme lactose permease. And lactose permease helps the cell bring lactose into the cell. And the lac a gene also codes for an enzyme that helps in lactose metabolism. We just won't focus on it because it's not as important as the lac z and lac y gene. So, these genes are all needed for the metabolism of lactose and let's just label them, this part over here, right before these three genes, that's the start site. So, if RNA polymerase was transcribing, that start site tells it here's where you should begin to transcribe and then after the lac a gene we have a stop region so that tells RNA polymerase, stop transcribing. And normally, e. coli uses glucose as its energy source. That's the default. However, if glucose is not available or if it's suddenly inundated with lactose it will want to break down lactose. But, why should e. coli express lac z, lac y, and lac a in the absence of lactose, right? We just explained before that would be a huge waste of energy. So, the default situation is that these genes are not expressed. Let's see how that is. So over here we had a promoter site and the promoter site is the place that the RNA polymerase kind of just sits. So let's label our RNA polymerase. That's the enzyme that puts together RNA. So it just sits there. And after the promoter site, we have the operator site. And on the operator site, there is a protein that also just sits there, and this is called a repressor. And you can see the repressor is kind of blocking the RNA polymerase, so normally, the RNA polymerase would want to proceed in this direction and transcribe this entire area of the DNA, but the repressor doesn't allow it to do that. It just sits there and blocks transcription. And so, again, this is the default situation that's in the cell. It uses glucose as an energy source and transcription of these genes is blocked. Well, what happens when there's suddenly a lot of lactose in the cell? So let's just draw some lactose molecules. I'm gonna draw them as these little triangles although that does not adequately represent what lactose looks like, but for now it'll, we'll just draw it like that. So they have a bunch of lactose floating in the cell. So what's going to happen is that a lactose molecule will attach itself to the repressor protein. This changes the confirmation of the repressor protein somewhat. And that causes the repressor to come off the operator site. So let's just get rid of our repressor. And let's put it, well, over here, for now. Together with the lactose that was attached to it. Now, the path of RNA polymerase is open, so it moves in this direction and transcribes all these genes. We make beta galactosidase, we make lactose permease and we have all the enzymes that we need to metabolize lactose. Well, what happens when the level of lactose goes down and we broke down all of our lactose? So let's get rid of some of our lactoses. Well, now, well, they're all taken care of including this lactose that was attached to the repressor and when the lactose comes off of the repressor, it changes the confirmation of the repressor and causes it to go back onto the operator site. So let's put him back where he was. Now, you can see the RNA polymerase again is blocked and so there is no more transcription happening right over here. So let's just connect this to what we spoke about in the beginning. We said that the regulation of gene expression happens at the level of transcription. We only transcribe those genes that we need and this is exactly what's happening over here. In the absence of lactose, these genes are not expressed and receiving the energy. But when we have lactose around, these genes are expressed and we have the enzymes necessary for the metabolism of lactose. |
48 | Biomolecules | Gene Control | Jacob-Monod: The Lac operon | So the JacobMonod model for gene expression describes the very first genetic regulatory mechanism to be understood clearly. When it was first described by French biologist Francois Jacob and Jacques Monad, who originated the idea that the control of enzyme levels in cells occurs through the regulation of transcription. And they, along with another scientist, shared the 1965 Nobel Prize in Medicine for their work on what is called the Lac Operon. Now a little bit of background first: An operon is a unit of genomic DNA containing a cluster of genes that are under control of a single regulatory signal, otherwise known as a promoter. And these genes are cotranscribed into a single mRNA strand and either translated together or undergo transsplicing to create single mRNA's that are translated separately. So, basically, genes in an operon are expressed either altogether or not at all. Now, the operon that I've drawn here happens to represent the lac operon, and the lac operon is an example of an inducible set of genes which are responsible for importing and breaking down the sugar molecule lactose to use as a source of energy. So, in the event that glucose, which is the ideal source of carbon and energy for a cell, if that's not available then the cell has sort of a backup source of energy in the form of lactose. And you can see where the name lac operon comes from because it is named for the inducer molecule for the operon. And what do I mean by inducer molecule? Well, it is the presence of lactose that actually induces the transcription of the genes in this lac operon, which I'll explain in just a little bit. So, there are three coordinately regulated genes contained in the lac operon. You have the lacZ gene, which codes for an enzyme called betagalactosidase, which is a cytoplasmic enzyme that cleaves lactoce into glucose and galactose. The next gene is the lacY gene, which codes for lactose permease, which is a cytoplasmic membrane protein that transports lactose into the cell. And then finally you have the lacA gene, which codes for thiogalactoside transacetylase. Now only the lacZ and the lacY gene are actually needed for lactose catabolism. LacA is not as important in terms of understanding how the lac operon works. Now besides these three structural genes, lacZ, Y, and A, there are two regulatory sequences contained in the lac operon, and they are called the promoter, which promotes the transcription structural genes if you will, and then also the operator. And there are two other regulatory sequences that lie just upstream of the lac operon that are genes that encode for a repressor protein, and then you have the associated promoter for that repressor protein. So, these are the structural genes here, and then here are the regulatory genes. Now, when glucose is readily available to the cell, the repressor protein is constitutively expressed, meaning that it is transcribed at base line and that is just the default. And this regulatory protein binds to the lac operator, and this interferes with and represses the binding of RNA polymerase which wants to bind here to the lac promoter. And this prevents and represses the transcription of these genes for lactose metabolism. Now, when glucose is not readily available to the cell, and an alternate source of energy is available in the form of lactose, then things start to change. First, lactose passively enters the cell at a pretty slow rate, and the metabolite of lactose, called allolactose, then binds to the repressor, and this alters the confirmation of this repressor protein, or it's shape, and it causes it to sort of loosen up and fall off the operator. Now, remember that the RNA polymerase is bound to the promoter immediately upstream of the genes. With the repressor now gone, RNA polymerase is free to sort of, picture it rolling down to transcribe all the three genes, leading to higher levels of the encoded proteins. So then you have lactose permease, which allows more lactose to enter the cell, and then you have more betagalactosidase which can break down the lactose into galactose and glucose to be used for the cells basic metabolic needs. Now, what happens if both glucose and lactose are present? Which one does the cell prefer? Well, in that case, the transport of glucose actually blocks the transport of the inducer, the lac operon, the lactose, in a process that's called inducer exclusion. So, actually the transport of glucose into the cell leads to the formation of this protein intermediate that binds to the lactose permease and prevents it from bringing in any more lactose into the cell. Then you have decreased lactose, which leads to decreased repressor protein bindings, so then the repressor protein then sort of latches back onto the operator there and prevents the transcription of the rest of the lac operon genes. Now, there are two key take away points from the lac operon model. The first is to realize that it is the interaction between the inducer and the repressor molecules that mediate gene expression. And the second idea is that the cell expends energy to make enzymes only when necessary. So, there are inducible genes whose transcription is induced when a particular molecule is present. |
49 | Biomolecules | Gene Control | DNA and chromatin regulation | So, the regulation of gene expression can be modulated at virtually any step in the process, from the initiation of transcription all the way to posttranslational modification of a protein, and every step in between. And it's the ability to regulate all these different steps that helps the cell to have the versatility and the adaptability of an efficient ninja, so that it expends energy to express the appropriate proteins only when needed. Or, you can think of the cell as a lazy couch potato that wants to expend the least amount of energy as possible. So, starting at the beginning of gene expression, let's talk about gene regulation as it pertains to DNA and chromatin regulation. Let's talk about the structure of DNA. DNA is packed into chromosomes in the form of chromatin, also know as supercoiled DNA. And so, chromatin is made up of DNA, histone proteins, and nonhistone proteins. And there are repeating units in chromatin, called nucleosomes, which are made up of 146 base pairs of double helical DNA that is wrapped around a core of eight histones. And there are four different types of histones within this structure of eight that you should be aware of. And they're named H2A, H2B, H3, and H4, that's just the nomenclature they've been given. Now, acetylation occurs at the amino terminal tails of these histone proteins by an enzyme called histone acetyltransferase, which I'll just abbreviate as HAT. And this is a reversible modification, so the acetylation of histones is sort of kept in balance by another enzyme that removes these acetyl groups, which is called histone deacetylase, or HDAC. The acetylation of histones leads to uncoiling of this chromatin structure, and this allows it be accessed by transcriptional machinery for the expression of genes. On the flip side of this, histone deacetylation leads to a condensed, or closed structure of the chromatin, and less transcription of those genes. When these modifications that regulate gene expression are inheritable, they are referred to as epigenetic regulation. So, when it comes to gene expression and DNA, you can basically think of DNA as coming in two flavors, densely packed, and transcriptionally inactive DNA, which is called heterochromatin, and then less dense, transcriptionally active DNA, which is euchromatin. And I like to think of heterochromatin as being densely packed and hibernating, like heterochromatin and hibernating both begin with H, kind of like a bunch of densely packed bears that are closed off in their cave for the winter, whereas euchromatin is waiting there with open arms, welcoming the transcriptional machinery to transcribe away. Now often you will see that histone deacetylation is combined with another type of DNA regulatory mechanism, and that is DNA methylation, and this occurs in a process called gene silencing. And this is a more permanent method of sort of downregulating the transcription of genes. And DNA methylation involves the addition of a methyl group, which is a carbon with three hydrogens, to the cytosine, DNA nucleotides, by an enzyme appropriately called methyltransferase. And this typically occurs in cytosinerich sequences called CpG islands. Don't forget that cytosine pairs with g, guanine, so that's why they're cg islands that you'll find. DNA methylation stably alters the expression of genes, and so it occurs as cells divide and differentiate from embryonic stem cells into specific tissues. And so this is essential for normal development, and is associated with other processes, such as genomic imprinting, and xchromosome inactivation, topics for another discussion. And abnormal DNA methylation has been implicated in carcinogenesis, or the development of cancer, so you can see how the normal functioning of DNA methylation is a critical regulatory mechanism for our cells. Now, DNA methylation may effect the transcription of genes in two ways. First, the methylation of DNA itself may physically impede the binding of transcriptional proteins to the gene. And second, and likely more important, methylated DNA may be bound by proteins known as methyl cpgbinding domain proteins, or MBDs, for short. Now MBD proteins can then recruit additional proteins to the locus, or particular location in a chromosome, certain genes, such as histone deacetylases, and other chromatin remodeling proteins, and this modifies the histones, forming condensed, inactive heterochromatin that is basically transcriptionally silent. |
50 | Biomolecules | Gene Control | Regulation of transcription | So what makes a cell that's located inside of your nose responsible for smelling, say, a slice of pizza look and act differently from a cell that lines your gut and is responsible for absorbing the nutrients from that pizza? They have the exact same DNA so the differences can't be attributed to that fact alone. The answer actually lies in the expression of that DNA, which genes are actively transcribed and which ones aren't and there are several ways in which gene regulation occurs at the level of transcription and so we're going to be talking about the main ones here. Now let's draw out a hypothetical gene here and associated with this gene is a sequence upstream so towards the three prime region of the antisense strand, also called the template strand. And this sequence is called the promoter and there is another sequence in between the promoter and the gene called the operator. The operator is the sequence of DNA to which a transcription factor protein combined and the promoter is the sequence of DNA to which the RNA polymerase binds to start transcription. Now first off in prokaryotes we have what are called general transcription factors, which are a class of proteins that bind to specific sites on DNA to activate transcription. General transcription factors plus RNA polymerase and another protein complex called the mediator multiple protein complex constitute the basic transcriptional apparatus, which positions RNA polymerase right at the start of a protein coding sequence or a gene and then releases the polymerase to transcribe the messenger RNA from that DNA template. Now there's another type of DNA binding protein called activators and these enhance the interaction between RNA polymerase and a particular promoter, encouraging the expression of the gene and activators can do this by increasing the attraction of RNA polymerase for the promoter through interactions with sub units of the RNA polymerase or indirectly by changing the structure of the DNA. An example of an activator is the catabolite activator protein or CAP and this protein activates transcription of the lac operon in E. coli. In the case of the lac operaon and E. coli, cyclic adenosine monophosphate or cAMP is produced during glucose starvation and so this cAMP actually binds to the catabolite activator protein or CAP which causes a confirmational change that allows the CAP protein to bind to a DNA site located adjacent to the promoter and then this activator, the CAP, then makes a direct protein to protein interaction with RNA polymerase that recruits the RNA polymerase to the promoter. Now enhancers are sites on the DNA that are bound to by activators in order to loop the DNA in a certain way that brings a specific promoter to the initiation complex and as the name implies this enhances transcription of the genes in a particular gene cluster. And while enhancers are usually what are called cisacting, cis meaning the same or acting on the same chromosome, an enhancer doesn't necessarily need to be particularly close to the gene that it acts on and sometimes it's not even located on the same chromosome. Enhancers don't act on the promoter region itself, but are actually bound by activator proteins and these activator proteins can interact with that mediator complex I mentioned earlier which recruits RNA polymerase and the general transcription factors which then can lead to transcription of the genes. So here I've drawn a little schematic of what it might look like to have the enhancer actually change the structure of the DNA so that the DNA is now looping around. Here you still have your promoter sequence, the operator sequence, the gene sequence, and the enhancer sequence, and having the DNA looped in such a way so that you could then recruit RNA polymerase, the transcription factors, the mediator protein complex, and then you have transcription begin of this gene here. Now let's talk about repressors. Repressors are proteins that bind to the operator, impending RNA polymerase progress on the strand and thus impeding the expression of the gene. Now if an inducer, which is a molecule that initiates gene expression, is present, then it can actually interact with the repressor protein in such a way that causes it to detach from the operator and then this frees up RNA polymerase to then transcribe the gene further down on the DNA strand. One example of a repressor protein is the repressor protein associated again with the lac operon operator, which prevents the transcription of genes used in lactose metabolism unless lactose, which is the inducer molecule, is present as an alternative energy source. Now silencers are regions of DNA that are bound by repressor proteins in order to silence gene expression and this mechanism is very similar to that of the enhancer sequences that I just talked about. And similarly, silencers can be located several bases upstream or downstream from the actual promoter of the gene and when a repressor protein binds to the silencer region of the DNA, RNA polymerase is prevented from binding to the promoter region. Now a few notes about the differences between prokaryotes and eukaryotes when it comes to transcriptional regulation. In prokaryotes, the regulation of transcription is really needed for the cell to be able to quickly adapt to the everchanging outer environment that it is sitting in. The presence, the quantity, the type of nutrients actually determines which genes are expressed and in order to do that, genes must be regulated in some sort of fashion so a combination of activators, repressors, and rarely enhancers, at least in the case of prokaryotes, determines whether a gene is transcribed. In eukaryotes, transcriptional regulation tends to involve a combination of interactions between several transcription factors which allows for a more sophisticated response to multiple conditions in the environment. And another major difference between eukaryotes and prokaryotes is the fact that eukaryotes have a nuclear envelope which prevents the simultaneous transcription and translation of a particular gene and this adds an extra spacial and temporal control of gene expression. |
51 | Biomolecules | Gene Control | Post-transcriptional regulation | Let's talk about posttranscriptional regulation which is regulation basically once DNA has been transcribed into mRNA and I've drawn out this little schematic for you here and it kind of just shows you how a DNA strand has a corresponding RNA strand and then the mRNA strand afterwards and I'll sort of explain what all the different colors and words mean in just a little bit. So once DNA is transcribed by RNA polymerase into the corresponding RNA strand, this RNA strand needs to get what I call a tidy little haircut and then don some protective outerwear before it can leave the comfort of the nucleus for its big debut into the cytoplasm in the form of a fully processed messenger RNA or mRNA strand. Now keep in mind that this form of regulation occurs in eukaryotes only and this modification also helps to stabilize the mRNA to protect it from premature degradation before it gets translated into a protein. So as you can see here, DNA gets transcribed one to one, base for base, into RNA and you can see here that there are sections of the RNA that ultimately make it into the finished mRNA, these short segments, which are turned exons, and they are the sequences that code for the ultimate protein product. And then there are short noncoding segments of RNA that get cut or spliced out and this would be the haircut that I alluded to earlier and so these are called introns, and this is accomplished by a large molecular entity called the spliceosome. So the spliceosome binds on either side of an intron, loops the intron into a circle, and then cleaves it off, and then ligates the two cut ends of the exposing exons together, kind of cinches them together. And an easy way to remember which sequences are exons, which ones are introns, is that exons exits the nucleus and introns stay in the nucleus so exons kind of stands for exit. Now even though the mRNA has gotten its nice little haircut, it's not quite ready to leave the nucleus just yet. It has to grab what is called a 5' prime cap and a 3' prime polyA tail. Now what are those things that I just mentioned? So a 5' prime cap refers to changes at the 5' prime end of the mRNA and remember that this is the phosphate end of the nucleotide bases in the mRNA and some people like to remember this as F for five prime and for fosphate so that's how you can kind of keep the two ends straight. And capping at the 5' prime end converts this end of the mRNA to a 3' prime end by a 5' prime to 5' prime linkage which basically just protects the mRNA from exonucleases which degrade foreign RNA. The cap also promotes ribosomal binding for translation and also helps the regulation of nuclear export of the mRNA. Now the polyA tail, that goes on the other end, the 3' prime end of the mRNA which has the terminal hydroxyl group and so what do I mean when I say polyA tail? Well, polyA tail refers to polyadenylation in which multiple adenosine monophosphates or basically adeonine bases are added to act as a buffer for exonucleases in order to increase the half life of mRNA and again, protect it from degradation. And so the purpose of the polyA tail is really very similar to the 5' prime cap which is basically to protect from degradation, help with promoting translation, and regulating nuclear export. The polyA tail also does one more thing and it kind of just helps with transcription termination for the RNA polymerase that's transcribing the messenger RNA. Polyadenylation is catalyzed by an enzyme called polyadenylate polymerase which as the adenosine monophosphates using adenosine triphosphate as the substrate and the polyA tail is built until it's about 250 or so nucleotides long. So overall the 5' prime cap and the polyA tail help to stabilize the mRNA for translation. That's the key point to take home from here. So once the mRNA has donned its cap and tail and had its introns spliced out, its now ready to exit the nucleus to be translated into a protein. Now additionally, there's one more type of RNA regulation called RNA editing, which is a process that results in seqeuence variation in the RNA molecule and is catalyzed by various enzymes. RNA editing is relatively rare and these events may include insertion, deletion, and base substitution of nucleotides within the edited RNA molecule. Now one of these enzymes is called adenosine deaminase acting on RNA or ADAR enzymes which convert specific adenosine residues to inosine in an mRNA molecule by hydrolytic deamination. Another type of editing is called cytosine deaminase acting on RNA, or CDAR, which involves deamination of cytosine to uridine by cytidine deaminase. RNA editing is currently being extensively studied in relation to infectious diseases because the editing process alters viral enzymes and their function so kind of an exciting, new emerging concept in posttranscriptional regulation. |
52 | Biomolecules | Gene Control | Non-coding RNA (ncRNA) | What is a noncoding RNA? A noncoding RNA, or an ncRNA, as it is abbreviated, is a functional RNA molecule that actually skips this last step and is not translated into a protein. In other words, they just go directly from transcription into an RNA molecule and then go off to perform any number of vital functions within the cell. There are many examples of noncoding RNAs, including micro RNAs, ribosomal RNAs, transfer RNA, the list goes on and on. As we go through each of these different types and examples of noncoding RNAs, you'll start to see that there's sort of an emerging theme, here. That is that most of these noncoding RNAs participate in either transcription or translation in one capacity or another. Let's start off with micro RNAs. Micro RNAs, or miRNAs, function in transcriptional and post transcriptional regulation of gene expression. They do this by base paring with complementary sequences within mRNA, or messenger RNA, molecules. This usually results in gene silencing through translational repression or target degradation. In essence, the mRNA to which these micro RNAs bind are prevented from being translated or they are sent on a pathway for degradation. The next set of noncoding RNAs that we'll be talking about are all involved in translation. The first of which is ribosomal RNA. Ribosomes are the cellular machinery used to translate mRNA into proteins. It is made up of one type of RNA molecule, ribosomal RNA. Transfer RNAs are an adapter molecule that links the codons in an mRNA strand to the corresponding amino acids. This is another type of noncoding RNA that you'll see in translation. The thrid type is called snow RNA, which stands for small nucleolar RNA. It's a class of small RNA molecules that guide covalent modifcations of ribosomal RNA, transfer RNA, and small nuclear RNAs, primarily through methylation, which is the addition of methyl groups, or pseudouridylation, which is the addition of an isomer of the nucleoside uridine. Another class of noncoding RNAs are the small nuclear RNAs, or snRNAs, not to be confused with the snow RNAs, the small nucleolar RNAs that we just talked about. Small nuclear RNAs get their name from the fact that the average length of these RNA molecules is approximately 150 nucleotides. Their primary function is in the processing of premRNA in the nucleus. They also aid in the regulation of transcription factors or a particular RNA polymerase, RNA polymerase two, as well as maintaining telomeres, which are the regions of repetitive nucleotide sequences at the end of a chromotid, which protects the end of the chromosome from deterioration during chromosomal replication. SnRNA can be associated with a set of specific proteins and form complexes that are called small nuclear ribonucleic proteins, or snRNPs or sometimes people just call them snRPs. There is a special snRP complex called the spliceosome, made up of five small nuclear RNAs and over 150 proteins that is responsible for splicing, or removing, the introns contained in messenger RNA, which is a major step in the post transcriptional modification that takes place in the nucleus of eukaryotes. The way the the spiceosome does this is that it binds to specific sequences in the premessenger RNA strand and performs two sequential transesterification reactions that splice out the intron and then [lagate] the two exons to form a mature mRNA. Now you know a little bit more some examples of noncoding RNAs and some of the functions that they perform within the cell. |
53 | Biomolecules | Gene Control | Oncogenes | Oncogenes are genes that code for proteins that normally direct cell growth. They start out as protooncogenes and then something happens to convert that protooncogene into a full blown oncogene and that's in some sort of tumorinducing agent or it could also be totally spontaneous. Now protooncogenes code for proteins that help to regulate cell growth and differentation which makes sense since the essence of a tumor, whether it is a cancerous or a benign one, is unregulated cell growth. In fact, the words oncogene and oncology, which is the study of cancer, share the same root word, onkos, which is Greek for mass or bulk. Now the products of these genes are often involved in signal transduction and the execution of mitogenic signals. And you should recall that a mitogen is a chemical substance that encourages a cell to start cell division, basically something that triggers mitosis. So how does a protooncogene make that switch into an oncogene? Well, there are three main mechanisms, deletion or point mutation, gene amplification or increased mRNA stability, and chromosomal rearrangement. So let's start with the first one. Deletion or point mutation in coding sequence of the DNA in the gene itself or within a regulatory region such as a promoter region can lead to either a protein that is produced in the same normal amounts but is hyperactive for some reason or maybe there's some sort of loss of regulation and the normal protein is just overexpressed. Gene amplification or an increase in mRNA stability that prolongs the existence of the mRNA and thus its activity in the cell can lead to a normal protein that is overexpressed. And then finally, chromosomal rearrangement involves translocation of a gene to a nearby regulatory sequence that then causes this normal protein, the gene product, to be overexpressed or you could possibly have fusion to an actively transcribed gene which overexpresses the fusion protein or leads to a hyperactive fusion protein. And so really you can start to see a theme here emerging where the key idea is that you have either a normal protein that is just overexpressed, basically too much of the normal protein or you can have normal expression but the protein itself is just a hyperactive one. Now there are tons of examples of oncogenes including the SRC oncogene, RAS, MYC, receptor tyrosine kinase, and also cytoplasmic tyrosine kinases. So let's talk about each one of those here. SRC was the first confirmed oncogene which was discovered in 1970, and was termed SRC for sarcoma, which is a tumor of mesenchymal cells or connective tissue cells. It was actually an oncogene discovered in a chicken retrovirus and so SRC codes for a nonreceptor protein tyrosine kinase so it phosphorylates specific tyrosine residues and other proteins. Another example of an oncogene is the RAS oncogene, which codes for a small GTPase which hydrolyzes GTP into GDP and phosphate. This protein is activated by growth factor signaling and functions like a binary switch, sort of an on/off switch, in growth signaling pathways. Examples of downstream effectors of RAS includes the protein MAPK, which is a type of kinase that in turn regulates genes that mediate cell proliferation. You can see examples of RAS oncogene mutations In thyroid tumors, certain leukemias, and cancers of the pancreas and colon. The MYC oncogene codes for a transcription factor that induces cell proliferation and there's a very common translocation involving MYC between chromosomes 8, where the MYC oncogene is found, and 14, which leads to a certain type of lymphoma called Burkitt's lymphoma. The RTK oncogene stands for receptor tyrosine kinase, which is responsible for adding phosphate groups to other proteins to turn them on or off. These are similar to the SRC gene which is a nonreceptor tyrosine kinase, the difference being the location of the protein, either at the cell surface as a receptor or not as a receptor. So say you have a cell surface receptor that receives a signal from outside of the cell. Well, then that signal gets propagated or transmitted Into the cell via these receptor tyrosine kinases that add phosphate groups to the target protein, specifically on tyrosine residues and they can cause cancer by turning a receptor constitutively or permanently on in the absence of signals from outside the cell. Some well known examples of receptor tyrosine kinases are vascular endothelial growth factor or VEGF, epidermal growth factor or EGFR, and platelet derived growth factor, PDGF. And finally we have the cytoplasmic tyrosine kinases, which mediate responses to the activation of receptors of cell proliferation, migration, differentiation, and survival. A well known example is the BCRABL gene in chronic myelogenous leukemia or CML, also known as the Philadelphia chromosome. It is a fusion of parts of DNA from chromosome 22 and chromosome 9. So basically a part of chromosome 22 which contains the BCR gene fuses with a fragment from chromosome 9 which contains the ABL gene. When these two chromosome fragments fuse, the genes also fuse, creating this new gene, the BCRABL gene. The fused gene codes for a protein that displays high protein tyrosine kinase activity, which is actually due to the ABL half of the protein and the unregulated expression of this protein activates other proteins that are involved in cell cycle and cell division which causes a cell to grow and divide uncontrollably, basically becoming cancerous. So as a result, the Philadelphia chromosome is associated wtih chronic myelogenous leukemia, a certain type of leukemia as I mentioned before, as well as other forms of leukemias, which are cancers of white blood cells. |
54 | Biomolecules | Gene Control | Tumor suppressors | Tumor suppressor genes are those genes whose protein products either have a halting effect on the regulation of the cell cycle, or they can also promote apoptosis, or sometimes both. So in other words these proteins are like big stop signs that act as safety checks to help stop the mistakes in cell division that can lead to uncontrolled cell growth and cancer. Now there are several sort of categories of tumor suppressor proteins. Those that recognize DNA damage and either repair it or initiate program cell death, apoptosis if it can't be repaired, so DNA repair proteins. Then there are those proteins that act as repressors of genes that are essential for the continuation of the cell cycle. So if these genes are actively repressed, and thus not expressed, the cell cycle does not continue on. So you have cell cycle repressors. With tumor suppressors there is this concept called the "TwoHit Hypothesis." In which both alleles, and remember that alleles are basically the copies for a certain gene. And you have two copies for any given gene. One on the chromosome you got from your mom. And one on the chromosome you got from your dad. Now in the TwoHit Hypothesis both alleles must be mutated before the effect is manifested. Because if only one of the alleles for the gene is damaged. Then you have this, sort of backup second copy, that can still produce the protective protein. So you need two hits. One hit for each of the alleles that you have. Another way that you can think of this is that in mutated oncogenes these alleles are typically dominant. So a mutation only one of the alleles yields the cancerous phenotype. But with a mutated tumor suppressor allele these mutations are recessive. Because both alleles must be mutated in order to lead to the cancerous phenotype. The TwoHit Hypothesis was first proposed with cases of Retinoblastoma. Rapidly developing cancer that originates from the immature cells of the retina. The light detecting tissue of your eye. And I'll write this as pRb for Retinoblastoma protein. Now the Retinoblastoma protein prevents the cell from replicating when its DNA is damaged. And it does this by preventing progression of the cell cycle from G1 into the S phase or synthesis phase. So the Retinoblastoma protein binds and inhibits transcription factors. Which normally push the cell into the S phase. And this complex acts as a growth suppressor and so the cell remains in the G1 phase. This complex also attracts a histone deacetylase protein to the chromatin. Which reduces transcription of S phase promoting factors. And you can remember this by recalling that histone deacetylase leads to chromatin condensation. Or transcriptionally inactive chromatin. So this also further suppresses DNA synthesis. Another very well known tumor suppressor protein is the p53 protein. Homozygous loss of this protein is found in up to 65% of colon cancers, 50% of lung cancers, and also in breast cancers. So this is clearly a very critical tumor suppressor protein. And so p53 activates DNA repair proteins when DNA has sustained damage. And it can also arrest growth by holding the cell cycle hostage, if you will, at the G1 to S regulation point. And this gives DNA repair proteins some time to fix the damage and allow for continuation of the cell cycle. So specifically p53 binds DNA and activates several genes including ones that code for protein called p21, whichs binds the cyclinCDK or cyclindependent kinase complex, which is actually the complex responsible for pushing the cell from the G1 to S phase in the cell cycle. P53 also functions in the initiation of apoptosis if the damage to DNA is irreparable. One significant exception to the TwoHit rule for tumor suppressor genes is with certain mutations of the p53 gene product. Which can then result in what is called a "Dominant Negative." Meaning that a mutated p53 protein can prevent the protein product of the normal allele from functioning. So don't forget to sort of keep that in the back of your mind when you're thinking about tumor suppressor genes. |
55 | Biomolecules | Genetic Mutations | An introduction to genetic mutations | Today I'm going to give you a quick introduction into genetic mutations. But first, let's go over the central dogma of molecular biology, which is just the idea that genetic information in a cell is formed in the form of DNA. This DNA is used to generate complimentary RNA through a process called transcription. That RNA is then used to synthesize a corresponding protein through the process of translation. Looking at a quick example, our short DNA strand here will be used to generate an RNA stand. Remember that A pairs with U or T and C pairs with G. Next, our RNA will be used to generate protein through translation. Remember that during this process, RNA nucleotides are read in groups of three, called codons, in order to generate corresponding amino acids. Just very generally, we say that mutations >>have the affect of making this synthesized protein not turn out quite right. I'm going to give a quick shoutout to sicklecell disease, which is an example of a disease that's caused by a genetic mutation. You may remember that there is a protein in red blood cells called hemoglobin, which we can also call Hb. Hemoglobin in a protein that coordinates to iron ions in order to hold onto oxygen molecules and transport them throughout the body. The mutation that causes sicklecell disease results in a mutated form of hemoglobin called HbS being formed, where the S is for the word sickle. The difference between normal hemoglobin and HbS is that one glutamate amino acid residue is being replaced with a valine amino acid residue. This small change results in all of these mutated HbS proteins aggregating together in a red blood cell, which makes it very difficult for that red blood cell to transport oxygen effectively. Just a side point, remember that red blood cells are initially generated from hematopoietic stem cells through a process called hematopoiesis. Where are mutations found, and how do they come up in the first place? Let's look at a couple of different possible mistakes that could lead to an incorrectly produced protein. First, we'll see what happens if a cell makes a mistake during translation. We'll stick with our example of sicklecell disease from before. Let's say that we have this sample piece of DNA with three nucleotides from the gene coding for hemoglobin. This DNA is transcribed to form the complimentary RNA sequence GAG. That GAG would normally correspond to a glutamate residue during translation, but a mistake during translation might lead to a valine residue being translated instead to produce the mutated hemoglobin associated with sicklecell disease. But notice that if a mutation happens during translation, the cell will only produce one mutated hemoglobin, or HbS, for each overall mistake. Since cells are making tons and tons of hemoglobin, just one mutated protein might not have that big of an effect on the cell. So we can say that mistakes during translation probably don't cause mutations like the one associated with sicklecell disease. Next, we'll look at mistakes during transcription. Again, we have our CTC piece of DNA, which would normally make GAG on RNA, but maybe a mistake occurs which leads to the transcription to a GUG instead, which would then code for the valine associated with mutated hemoglobin. If this mistake occurred, the cell would only make a few mutated hemoglobins for each mistake since an individual strand of messenger RNA will only be translated a couple of times before being degraded. >>So we can say that mistakes during transcription probably don't cause mutations like the one associated with sicklecell disease. Finally, we'll look at mistakes in the DNA strand. If our CTC in DNA is mistakenly turned into a CAC, then our corresponding RNA from transcription will be changed and ultimately a valine would be produced instead of a glutamic acid. Now, since a cell's DNA stores all of its genetic information, that mistake would lead to all future hemoglobins produced from that gene being mutated. So overall, we can say that mutations will usually result from mistakes in a cell's DNA and not from the RNA or the protein. So where do these types of mutations come from? There are two ways a person can get a genetic mutation. The first is that they inherit it from their parents. Remember that DNA is passed down from parents to offspring, so if we have a mutated father here, then there's a good chance that at least one of his kids will inherit that mutated gene the same way that the child might inherit any amount of that parent's DNA. The other possibility is that the mutation will come on spontaneously, which is where a person suddenly gets a mutation in their DNA without their parents having had the same mutation. Spontaneous mutations can come from many different sources, with just a few examples being from DNA replication errors, environmental factors like certain poisons. It's also possible that genetic mutations can come on entirely randomly. What did we learn? First we learned that mutations originate at the DNA level, and not the RNA or protein level. The effects of the mutation, like the example we gave of sicklecell disease, are found with problems with the proteins that are ultimately expressed by the mutated DNA. Now, like every rule, there are a couple of exceptions to this one, but we can say that the effects of a mutation are usually found at the protein level. Finally, we learned that mutations are either inherited from a parent, or come on entirely spontaneously. |
56 | Biomolecules | Genetic Mutations | The different types of mutations | So, today we're going to talk about the different types of genetic mutations that you would find in a cell. But first, I want to review the central dogma of molecular biology and how the genetic information of a cell is stored in the form of DNA, which is then transcribed to form RNA and then translated to generate protein. Nucleotides from the DNA are transcribed to their complementary forms on RNA, which are then read as codons or groups of three, to code for specific amino acids in a larger protein. Now, if you mutate one of the nucleotides on DNA, like let's say turning this thyminebased into an adeninebased, then that will affect the RNA sequence and ultimately the protein that follows. So, we say that mutations are mistakes in a cell's DNA that ultimately lead to abnormal protein production. So, what are the different types of mutations? Well, the first type of mutations we're going to talk about are called point mutations. Now, here I've just written out a random sequence of DNA, which is just a repeating pattern of CTC, which would code for a repeating sequence of GAG in the RNA strand, and finally, a protein sequence of three glutamate amino acids. So, a point mutation is when one of our DNA bases is replaced with another. So, in this example, a thyminebased is being replaced with an adeninebased, which leads to a change in one RNA nucleotide and ultimately a change in one amino acid. Another type of mutation is called frameshift, which works a little differently. So, first I'll write out the same DNA, RNA, and protein sequences from before, but now, instead of changing one base to another, I'm going to add one to the sequence, and here I've thrown in this extra cytosine base that I've written in blue. Now, naturally, this change would lead to an additional guanine base being in the resulting messenger RNA sequence, but what's interesting is that this mutation will change the reading frame of the RNA. Remember that RNA is read in groups of three or codons when being translated to form protein, but now, since we've added an extra G here, all of the codons coming after that extra G will look a little differently. Now, instead of having three GAG codons, we've swapped out two for GGA codons. This means that two of our amino acids in the final protein will be changed, and in this example, they'll be changed from glutamate to glycine. So, you can see that frameshift mutations usually have more significant effects on the final protein than point mutations do. Now, it's important to recognize that both of these mutations are classified and named for how they affect the cell's DNA structure and aren't really named for how they affect the resulting protein. Now, our next type of mutations are nonsense mutations and missense mutations. Let's say we have a DNA sequence that normally generates RNA and codes for a cysteine amino acid. A nonsense mutation is any genetic mutation that leads to the RNA sequence becoming a stop codon instead. Now, missense mutations are a little different, and they're any genetic mutation that changes an amino acid from one to another. So, in this example, our mutation is changing the resulting amino acid from a cysteine to a tryptophan. Now, you can see that nonsense mutations probably affect the resulting protein a lot more than missense mutations do, since that new stop codon that we're creating could chop off a huge section of the protein, instead of just changing one amino acid to another. So, now we can divide the missense mutations even further into a bunch of smaller categories. Silent mutations are when the mutation doesn't actually affect the protein at all. Since many different RNA codons can code for the same amino acid, it's possible that the mutation might not affect the protein at all. So, in this example, CCA, CCG, CCT, and CCC in the section of DNA will all end up coding for glycine. So, if you change the third base, it wouldn't affect the final protein. Conservative mutations are where the new amino acid is of the same type as the original. So, here I have a glutamate and an aspartate, which are both acidic amino acids. So, a mutation that swapped out an aspartate for a glutamate would be a conservative mutation. Finally, a nonconservative mutation is one with a new amino acid is of a different type from the original. So, here we have a serine amino acid, which is a small polar amino acid, being replaced with phenylalanine, which is a large, nonpolar, aromatic amino acid, and this would be an example of a nonconservative mutation, since serine and phenylalanine are different types of amino acids. Now, I'll point out again that all of these mutations are classified and named for how they affect the resulting proteins and aren't really named for how they affect the cell's DNA. So, let's look at a quick example. Sickle cell disease is a disorder where hemoglobin or Hb, which is a protein found in human blood, is mutated into a less active form, which we're going to call HbS, and it results from a single glutamate residue being converted into a valine residue. Now, we can classify this mutation as a point mutation, since only one DNA base is affected, but we can also say that it's a nonconservative missense mutation, since glutamate is being swapped out for valine, and the two are different types of amino acids, since glutamate is an acidic amino acid, and valine is a nonpolar one. So, what did we learn? Well, first we learned that mutations originate at the DNA level, but show their effects on the protein level, and second, we learned that we can classify different types of mutations by either their effects on DNA or their effects on protein. In reference to DNA, we have point and frameshift mutations, and in reference to protein, we have missense and nonsense mutations. |
57 | Biomolecules | Genetic Mutations | The causes of genetic mutations | So, today we're going to talk about the causes of genetic mutations, but first let's just do a quick review of the idea that mutations are mistakes in a cell's DNA, and there are two main types of mutations that we see when we look at a cell's DNA, and the first is called point mutations, and that's when one DNA base is switched out for another, which usually results in a change to one codon in the RNA sequence. Frameshift mutations are when the reading frame of the RNA is altered, and while the actual nucleotides in the RNA sequence haven't changed that much, the reading frame of the RNA strand has shifted, meaning that many different RNA codons will change as a result, and we're going to take a look into what causes these point and frameshift mutations. So, point mutations are caused by base substitution, which is when one DNA base is substituted for another, and there are a couple of different types of base substitution. A transition is when you have a substitution of adenine for guanine or vice versa, which is a swap between two purines, or a substitution of cytosine for thymine or also vice versa, which is a swap between two pyrimidines. A transversion is when either adenine or guanine is swapped for either cytosine or thymine, and in this type of base substitution, you have either a purine being replaced with a pyrimidine or a pyrimidine being replaced with a purine. Now, the last kind of mutation that can lead to a point mutation is a mispairing, which some people call mismatching, and that's when a DNA strand has a nonWatsonCrick base pairing. Normally, A pairs with T and G pairs with C, but when you have a mispairing, that's when A and C pair up or when G and T pair up, and it's much more common for mispairings to occur between a purine and pyrimidine, as opposed to between two purines, like A and G pairing up, or two pyrimidines like C and T pairing up. Next, we're going to talk about frameshift mutations. So, let's say that we have this DNA strand here, with three repeating CTC units and an extra C on the end. This would then be transcribed into an RNA strand with repeating GAG units and an extra G on the end, and our three codons would be the three GAG units, which would then each translate to a glutamate amino acid. Now, one way you can cause a frameshift mutation is through an insertion, and that's when an extra DNA base finds its way into our sequence. So, here we have this extra cytosine base, that I've underlined, falling into our sequence, and this additional C base would lead to an extra G being thrown into our RNA sequence, which would then shift the codon reading frame of our RNA strands during translation. So now, instead of three GAG codons, we have just one GAG codon and two GGA codons, with two extra bases on the end. This would then code for one glutamate residue and two glycine residues, instead of three glutamates. The other way that you can cause a frameshift mutation is through a base deletion. So, in a deletion, we drop off one of our bases from our original sequence. So, here I've dropped that first thymine base, and this would also result in a shift of the RNA reading frame. Now, instead of having three GAG codons, we have a GGG codon and two AGG codons, which would lead to a protein with a glycine and two arginine amino acids. So, overall, insertions and deletions can both lead to frameshift mutations. Now, we can also talk about largescale mutations, which instead of being at the level of individual nucleotides, are usually seen at the chromosomal level and can affect many genes, instead of just a few base pairs. So, first we'll talk about translocation, which is when a gene from one chromosome is swapped for another gene on a different chromosome. Now, it's important to see that translocation refers to gene swapping between nonhomologous chromosomes, which means that if this blue chromosome were chromosome 10, then the green one could be any chromosome aside from chromosome 10, and this is what sets translocation apart from the process of crossing over that occurs during meiosis between homologous chromosomes. The next largescale mutation we'll talk about is chromosomal inversion, and that's when two genes on the same chromosome switch places. So, here our green and blue genes are being swapped and end up on different parts of the chromosome after the mutation. Now, since both of these mutations don't always affect the individual nucleotides coding for a gene, it's important to see that many of these types of mutations affect how a gene's expression is regulated, in addition to changing what the genes actually code for. Remember that the position of a gene on a chromosome partly determines how it's regulated, and this could be due to histone configuration, promoter regions, or any other regulatory process. So, what did we learn? Well, first we learned that smallscale mutations affect the DNA at the nucleotide level, and of these smallscale mutations, we have point mutations, which can be caused by transitions, transversions, and mispairings, and we also have frameshift mutations, which can be caused by insertions or deletions. Next, we talked about largescale mutations, which affect the DNA at the chromosomal level, and the two largescale mutations we talked about were translocation and inversion. |
58 | Biomolecules | Genetic Mutations | Mutagens and carcinogens | So, today we're going to talk about mutagens and carcinogens and how they can cause DNA damage, but first I want to review the central dogma of molecular biology and how the genetic information of a cell is stored in the form of DNA, which is then transcribed to form RNA and then translated to generate protein. Now, nucleotides from DNA are transcribed to their complementary forms on RNA, which are then read as codons, or groups of three, to code for specific amino acids in a larger protein. Now, if you mutate one of the nucleotides on DNA, like turn this thymine base into an adenine base, then that will affect the RNA sequence and ultimately the protein that follows. So, we say that mutations are mistakes in a cell's DNA that ultimately lead to abnormal protein production. So, what is a mutagen? Well, a mutagen is any chemical substance or physical event that can cause genetic mutations. Chemical substances, like certain poisons, could be mutagens or physical events, like UV light or different kinds of radiation could also be mutagenic, and we classify mutagens into two different categories. So, let's say we have a person over here. A mutagen could be classified as endogenous, if it comes from inside this person's body, and it's some mutagen that's already found in the organism, but an exogenous mutation is one that comes from outside the affected organism, something that's from the external environment. So, what are some examples of some endogenous mutagens? Well, the most significant endogenous mutagens are what we call reactive oxygen species or ROS, and ROS are naturally occurring metabolites in the human body that are produced by mitochondria during oxidative phosphorylation. So, if we have this guy here, who's about to chow down on a big meal, you can expect that during the metabolism of the meal, his mitochondria will produce a bunch of ROS, like O2 dot minus, which we call superoxide, which is an oxygen molecule with one extra electron, as well as some hydrogen peroxide, which is another ROS that your body can produce. Now, reactive oxygen species, as you may be able to tell by their name, contain oxygen, like both of these examples do, but they're also highly reactive with different cell components, including DNA, and by reacting with DNA, they can actually cause significant damage to a cell's genetic code. One example of this type of damage is the doublestrand break, and ROS can actually break a DNA's double helix into two smaller pieces, and you can see why this type of a reaction could cause a mutation, since it quite significantly changes the structure of the cell's DNA. The next type of DNA damage that ROS can cause is base modification, and that's when the nucleic acid bases are changed or swapped around, and that can pretty readily cause point mutations or maybe even other kinds. Now, you may be wondering why would a cell ever make something that could damage itself? Well, it turns out that ROS actually have a couple of beneficial effects on a cell, and cells actually have a couple of ways to make sure that they don't cause damage, but sometimes ROS levels get really high, and cells can't deal with them anymore, and we call this oxidative stress, and antioxidants are something that your doctor might have told you that are good for you, and it turns out that part of what antioxidants do is help make sure that ROS don't damage your DNA. Now, let's look at a couple examples of exogenous mutagens, and there are many different types of exogenous mutagens, but we're really only going to talk about two. Now, intercalators are one example, and one of them is called ethidium bromide, which you may be familiar with if you've ever done a PCR experiment before, and what ethidium bromide will do is it will jump into a DNA double helix and stick itself between the two strands, and when these intercalators intercalate into DNA, they can deform the structure of the DNA and cause some serious problems. Base analogs, like 5bromouracil, which we also call 5BU, pretend to be a certain base, but then act differently than that base normally would. So, in the case of 5BU, it's an analog of uracil and looks a lot like it, but once it's incorporated into DNA, it can shift between two different forms. In its ketoform, it will pair best with adenine. While it's in enolform, it will pair best with guanine. Now, if you're familiar with organic chemistry, you might know that 5BU can convert between its keto and enol form through something called a tautomerization reaction, and overall you can see how this base analog might be able to induce mutations in a DNA strand. Now, the last thing we're going to talk about is what a carcinogen is. Now, carcinogens can be mutagens, but not all of them are, but in general, you can say that a carcinogen is something that can lead to cancer, which, if you remember, is when cells in an organism divide uncontrollably and can form big masses of cells, called tumors. Now, some carcinogens will work by making mutations in DNA that lead to cancer, but sometimes they might carry out their effects simply by increasing the rate at which a bunch of cells divide, without actually affecting their DNA, and some examples of carcinogens are tobacco, which come from cigarettes, asbestos, which used to be used as home insulation, and even UV radiation. So, what did we learn? Well, first we learned that mutagens are chemical or physical substances or events that can increase the probability of genetic mutations occurring. And next we learned that carcinogens are things that lead to cancer, and while they can be mutagenic as well, they aren't necessarily mutagenic. |
59 | Biomolecules | Genetic Mutations | The effects of mutations | so today we're going to talk about the overall effects of a genetic mutation and how mutations impacts the affected organism as a whole but first I want to review the central dogma of molecular biology and how genetic information in a cell is stored in the form of DNA which is then transcribed to form RNA which is then translated to form protein now nucleotides from DNA are transcribed to their complimentary forms on RNA which are then read as codons or groups of three to code for specific amino acids in a larger protein now if you mutate one of the nucleotides on DNA lecturing a thymine base into an adenine base then that will affect the RNA sequence and ultimately the protein that follows so we say that mutations are generally mistakes in a cell's DNA that lead to abnormal protein production so our mutations good or are they bad and what kind of a factor they have on the affected organism well there isn't really a good answer to this question at all and there are many many different types of mutations out there that can result in big structural changes like the little pictures I've drawn out here or may result in little subtle changes that might go completely unnoticed it's very difficult to call a mutation good or bad though since it really depends on a huge number of things including the environment that the organism lives in so let's look at an example of a good mutation so the bacteria streptococcus pneumoniae is the bacteria that you typically see associated with pneumonia and one of the more popular treatments for pneumonia is giving the infected person an antibiotic like penicillin which would help kill all of the bacteria and get rid of the disease but sometimes you can find some mutated streptococcus bacteria that will have a special trait that makes them resistant to penicillin and now penicillin won't kill them as easily as it will kill the bacteria without the mutation now we call this a good mutation because the bacteria are living in a human host where they're likely to encounter this deadly penicillin and being resistant to antibiotics like penicillin would then be beneficial to the bacteria and just to clarify I'm calling this a good mutation for the bacteria not really for the human infected since it'll be harder for them to get rid of the bacteria that are resistant to certain so now let's look at an example of a bad mutation so the disease cystic fibrosis is usually caused by a mutation in the CFTR gene now I'm not really going to go into detail about how this mutation actually hurts you but I'll leave you with the idea that what it does is it makes the mucus that you'd find in a person's lungs really really thick which makes it really hard for people affected with the disease to breathe so in general we can say that the de mutation causing cystic fibrosis would be a quote unquote bad mutation but mutations aren't strictly good or strictly bad in fact there are some mutations that can cause some favorable and some disadvantageous effects sicklecell disease results from a mutation in a protein called hemoglobin that you'd find in red blood cells and this mutation terms hemoglobin into a much less functional form which we'll call HBS and it's much less efficient at moving oxygen around the human body but another effect of sicklecell disease is that it makes the disease person less susceptible to malaria now malaria is a parasite that grows and multiplies in red blood cells and can have a lot of nasty effects on the host organism and the malaria parasite can't really grow as well in red blood cells that are affected with sicklecell disease so in this case the mutation associated this disease has one bad effect which is that the HBS isn't as good as carrying oxygen around the body but also a good effect in that it makes it less likely that the disease person will be affected by malaria since they can't grow as well in the humans red blood cells so what did we learn well first we learn that the effects of the mutation will usually but not always appear at the protein level there are some exceptions to this rule and second we learn that genetic mutations can have advantageous deleterious or neutral effects depending on the type of mutation the environment that the affected organism lives in as well as a multitude of other factors |
60 | Biomolecules | Mendelian Genetics | An Introduction to Mendelian Genetics | [Voiceover] An introduction to Mendelian Genetics. Now before we start, let's review the idea that human cells contain 46 chromosomes, which contain the DNA that makes each cell unique. 23 of these chromosomes were inherited from a person's father and 23 were inherited from the mother. We can say that each person's made up of a combination of genetic code from both of their parents. Now sometimes we like to say that we have 23 pairs of chromosomes. Instead of saying that we have 46 total because that way we remind ourselves that for each chromosome we have a maternal and paternal copy. Now the first thing I want to introduce is the term allele. If we have a chromosome here and then an allele is one small section on that chromosome that codes for a specific gene that makes you, you. Since humans have at least two copies of each chromosome, we can say that humans usually have at least two alleles for every specific gene. One allele from their mother and one from their father. Let's look at an example and we'll start by talking about blood type. I'm sure that you've heard that blood types are usually named with letters like A, B, and O. What does that actually mean? Well there's a specific allele that codes for blood type. Let's say that we have this guy here and his alleles both code for blood type A. I'll use the letter A for that. Let's say we have this girl here who has one allele coding for A and another allele coding for blood type O. Now for the guy, he has both alleles coding for blood type A then it's pretty clear that when we check his actual blood type it will be A. For the girl, we're not so sure since she has one of each. Now, I'm going to introduce a couple new terms to you. The first is that since the guy has two alleles that code with the same thing both code for blood type A then we say that this guy is homozygous. Homo means the two alleles are the same, homo the same and zygous refers to mixture of DNA that he got from his parents. Someone who is homozygous got the same allele from both parents. In the case of the girl, is she going to have blood type A or blood type O? Well it turns out that she's going to have blood type A and that's because the A allele is the dominant allele. While the O allele is the recessive allele. When an allele is dominant that means if someone has two different alleles it will be the dominant one that wins. In this case since A is dominant over O which is recessive, A will win and she'll have blood type A. Since this girl has two different alleles we call her heterozygous since hetero means different and zygous refers to the same thing, a mixture of DNA that she got from her parents. Now I want to introduce two more terms. We can describe a person's genes in two different ways. We can look at the person's individual alleles and we call this the genotype. For this guy his genotype is AA referring to his two alleles which both code for blood type A. We can also look at a person's physical traits which we call the phenotype. For this guy and girl the phenotype would be blood type A. You can see that genotype and phenotype are different but it is possible for two different genotypes to make the same phenotype. Since some alleles are dominant over others. Let's talk about gene inheritance for a bit. Let's say that our guy and girl from before have offspring together. We can use something called a Punnett Square to determine what different genotypes their kids could have. Each of the parents two alleles are on separate chromosomes, so each parent will contribute one of their two alleles to the child. The Punnett Square allows you to determine all possible combinations. If we take the father's alleles and line them up vertically and then take the mother's alleles and line them up horizontally, we can fill in the chart to find the possible genotypes for our offspring. In this case, two of our boxes will have the AA in them and two will have AO in them. That means half of the children will have the genotype AA and half of the children will have genotype AO. Since both of these genotypes code for the same phenotype all of the children will have the blood type A phenotype. Let's see what happens if we change our father's genotype to match our mother's genotype. Now only onequarter of the children will have the AA genotype, half will have the AO genotype since the order of the two alleles doesn't matter OA and AO are the same. One quarter will have the OO genotype. This means that 75% of the children will have blood type A in their phenotype. Since AA and AO make blood type A but 25% of the children will have the blood type O phenotype, since OO makes blood type O. What did we learn? Well first we learned what an allele is and the difference between homozygous and heterozygous, as well as the difference between dominant and recessive traits in relation to alleles. Second, we learned about the difference between genotype and phenotype and how the genotype refers to a persons DNA while a phenotype refers to the physical traits that the DNA codes for. Finally we learned about how we can use a Punnett Square to determine how different alleles will be inherited from two parents. |
61 | Biomolecules | Mendelian Genetics | Co-dominance and Incomplete Dominance | So today we're gonna talk about CoDominance and Incomplete Dominance, but first let's review the example of a blood type and how someone with the same two alleles coding for the same trait would be called homozygous and someone with different alleles would be called heterozygous. Also remember, the concept of dominant and recessive alleles and how the A allele is dominant over the O allele in this example. This means that the same phenotype, blood type A, can result from these two different genotypes. Now, the example that I just gave you was an example of Complete Dominance. So if a person had a genotype AO, since our phenotype is just blood type A, it means that the A allele is completely dominant over the O allele and only the A allele from the genotype is expressed in the phenotype. But there are actually three different patterns of dominance that I want you to be familiar with and to explain this I'm going to use a different example. Let's say we have this flower and the red petal phenotype is coded for by the red R allele and the blue flower phenotype is coded for by the blue R allele. So I'm going to introduce three different patterns of dominance and they are complete dominance, which you've already heard of, codominance, and also incomplete dominance. I'm going to explain what these two new patterns are through this flower example. Let's start by looking at three different genotypes and the phenotypes that you would see for each of them under each different dominance pattern. We'll start with the genotype, two red Rs, which we could expect that in all cases the flower petals will be red since we only have red Rs in the genotype. Similarly, if our genotype had two blue Rs then we could expect that in all cases the flower petals will be blue since we only have blue Rs in the genotype. Now these three different dominance patterns change when we look at the heterozygous example. That's what makes these three patterns different. Now we're already familiar with the example of complete dominance, so if we said that the red R is dominant over the blue R then this would make the heterozygous phenotype a red flower for complete dominance. Now what codominance is, is when the heterozygous phenotype shows a flower with some red petals and some blue petals. So it's when the two alleles are dominant together they are codominant and traits of both alleles show up in the phenotype. Now what incomplete dominance is, is when the heterozygous phenotype shows a mixture of the two alleles. So in this case the red and blue flower petals may combine to form a purple flower. Neither allele is completely dominant over the other and instead the two, being incompletely dominant, mix together. So what did we learn? Well, if we assume the heterozygous genotype, red R, blue R, then there are three different dominance patterns that we might see for a specific trait. In complete dominance, only one allele in the genotype, the dominant allele, is seen in the phenotype. And this was the example with the red flower. In codominance, both alleles in the genotype are seen in the phenotype. This was the example with the flower with both red and blue petals. Finally, in incomplete dominance, a mixture of the alleles in the genotype is seen in the phenotype and this was the example with the purple flower. |
62 | Biomolecules | Mendelian Genetics | Worked example: Punnett squares | In the last video, I drew this grid in order to understand better the different combinations of alleles I could get from my mom or my dad. And this grid that I drew is called a Punnett square. And I looked up what Punnett means, and it turns out, and this might be the biggest takeaway from this video, that when you go to the farmers' market or you go to the produce and you see those little baskets, you see those little baskets that often you'll see maybe strawberries or blueberries sitting in, they have this little grid here, right there. Sometimes grapes are in them, and you have a bunch of strawberries in them like that. That green basket is a punnett. That's a punnett. Apparently, in some countries, they call it a punnett. I think England's one of them, and you UK viewers can correct me if I'm wrong. And so I guess that's where the inspiration comes for calling these Punnett squares, that these are kind of these little green baskets that you can throw different combinations of genotypes in. And these Punnett squares aren't just useful. If you're talking about crossing two hybrids, this is called a monohybrid cross because you are crossing two hybrids for only one trait. It could be useful for a whole set of different types of crosses between two reproducing organisms. It doesn't even have to be a situation where one thing is dominating another. Let's do a bunch of these, just to make you familiar with the idea. So let's say you have a mom. So instead of doing two hybrids, let's say the mom I'll keep using the blueeyed, browneyed analogy just because we're already reasonably useful to it. Let's say that she's homozygous dominant. And let's say that the dad is a heterozygote, so he's got a brown and he's got a blue. And we want to know the different combinations of genotypes that one of their children might have. So what we do is we draw a Punnett square again. Let me draw a grid here and draw a grid right there. And up here, we'll write the different genes that mom can contribute, and here, we'll write the different genes that dad can contribute, or the different alleles. I didn't want to write gene. I wanted to write dad. So the mom in either case is either going to contribute this big B brown allele from one of the homologous chromosomes, or on the other homologous, well, they have the same allele so she's going to contribute that one to her child. The dad could contribute this one, that big browneyed the capital B allele for brown eyes or the lowercase b for blue eyes, either one. So the different combinations that might happen, an offspring could get both of these brown alleles from one copy from both parents. This could also happen where you get this brown allele from the dad and then the other brown allele from the mom, or you could get a brown allele from the mom and a blueeyed allele from the dad, or you could get the other browneyed allele from the mom, right? When the mom has this, she has two chromosomes, homologous chromosomes. Each of them have the same brown allele on them. They both have that same brown allele, so I could get the other one from my mom and still get this blueeyed allele from my dad. So if you said what's the probability of having a blueeyed child, assuming that blue eyes are recessive? And remember, this is a phenotype. These particular combinations are genotypes. Well, in order to have blue eyes, you have to be homozygous recessive. You have to have two lowercase b's. So what's the probability of having this? Well, there are no combinations that result in that, so there's a 0% probability of having two blueeyed children. What's the probability of having a homozygous dominant child? Let me write that. A homozygous dominant. And now we're looking at the genotype. We care about the specific alleles that that child inherits. Well, which of these are homozygous dominant? Well, you have this one right here and you have that one right there, and so two of the four equally likely combinations are homozygous dominant, so you have a 50% shot. And we can do these Punnett squares. They don't even have to be for situations where one trait is necessarily dominant on the other. For example, you could have the situation it's called incomplete dominance. Let's say you have two traits for color in a flower. You could have red flowers or you could have white flowers. And let's say I were to cross a parent flower that has the genotype capital R I'll just make it in a capital W. So that could be the mom or the dad, although the analogy breaks down a little bit with parents, although there is a male and female, although sometimes on the same plant. And let's say the other plant is also a red and white. The other plant has a red allele and also has a white allele. So what are the different possibilities? Well, we just draw our Punnett square again. Let me draw our little grid. So the child could inherit both of these red alleles. He could inherit this white allele and then this red allele, so this red one and then this white one, right? That's that right there and that red one is that right there. Or it could inherit this red one from let's say this is the mom plant and then the white allele from the dad plant, so that's that one right there. Or you could inherit both white alleles. What I said when I went into this, and I wrote it at the top right here, is we're studying a situation dealing with incomplete dominance. So what does that mean? Well, that means you might actually have mixing or blending of the traits when you actually look at them. So if this was complete dominance, if red was dominant to white, then you'd say, OK, all of these guys are going to be red and only this guy right here is going to be white, so you have a one in four probability to being white. But let's say that a heterozygous genotype so let me write that down. Let's say when you have one R allele and one white allele, that this doesn't result in red. This results in pink. So this is what blending is. It's kind of a mixture of the two. So if I said if these these two plants were to reproduce, and the traits for red and white petals, I guess we could say, are incomplete dominant, or incompletely dominant, or they blend, and if I were to say what's the probability of having a pink plant? And now when I'm talking about pink, this, of course, is a phenotype. So the probability of pink, well, let's look at the different combinations. How many of these are pink? This one is pink and this is pink. So two are pink of a total of four equally likely combinations, so it's a 50% chance that we're pink. And we could keep doing this over multiple generations, and say, oh, what happens in the second and third and the fourth generation? Actually, we could even have a situation where we have multiple different alleles, and I'll use almost a kind of a more realistic example. I'll use blood types as an example. So there's three potential alleles for blood type. You can have a blood type A, you could have a blood type B, or you could have a blood type O. What happens is you have a combination here between codominance and recessive genes. And I'm going to show you what I talk about when we do the Punnett squares. Maybe I'll stick to one color here because I think you're getting the idea. So let's say I have a parent who is AB. So that means that they have on one of their homologous chromosomes, they have the A allele, and on the other one, they have the B allele. That's what AB means. So the phenotype is the genotype. They're codominant. They both express themselves. They don't necessarily blend. They both express. That's an AB blood type. Let me write this right here. This is AB blood type. And then the other parent is let's say that they are fully an A blood type. Let's say they're an A blood type. Let's say their phenotype is an A blood type I hope I'm not confusing you but their genotype is that they have one allele that's an A and their other allele that's an O. So this is what's interesting about blood types. It's a mixture. O is recessive. O is recessive, while these guys are codominant. So if you have either of these guys with an O, these guys dominate. If you have them together, then your blood type is AB. So what are all the different combinations for these for this couple here? Well, you could get this A and that A, so you get an A from your mom and you get an A from your dad right there. And clearly in this case, your phenotype, you will have an A blood type in this situation. You could get the A from your dad and you could get the B from your mom, in which case you have an AB blood type. You could get the A from your mom and the O from your dad, in which case you have an A blood type because this dominates that. Or you could get the B from your I dont want to introduce arbitrary colors. You could get the B from your mom, that's this one, or the O from your dad. No, once again, I introduced a different color. And this is a B blood type. So if I said what's the probability of having an AA blood type? And once again, we're talking about a phenotype here. So which of these are an A blood type? This one definitely is, because it's AA. If you have two A alleles, you'll definitely have an A blood type, but you also have an A blood type phenotype if you have an A and then an O. O is recessive. So this is also going to be an A blood type. So these are both A blood, so there's a 50% chance, because two of the four combinations show us an A blood type. And you could do all of the different combinations. You say, well, how do you have an O blood type? Well, both of your parents will have to carry at least one O. So, for example, to have a that would've been possible if maybe instead of an AB, this right here was an O, then this combination would've been two O's right there. So hopefully, that gives you an idea of how a Punnett square can be useful, and it can even be useful when we're talking about more than one trait. So let's go to our situation that I talked about before where I said you have little b is equal to blue eyes, and we're assuming that that's recessive, and you have big B is equal to brown eyes, and we're assuming that this is dominant. And let's say we have another trait. I introduced that tooth trait before. So let's say little t is equal to small teeth. I don't know what type of bizarre organism I'm talking about, although I think I would fall into the big tooth camp. Let's say big T is equal to big teeth. So an individual can have for example, I might be heterozygous brown eyes, so my genotype might be heterozygous for brown eyes and then homozygous dominant for teeth. So this might be my genotype. And the phenotype for this one would be a bigtoothed, browneyed person, right? Let me make that clear. This is big tooth phenotype. And this is the phenotype. What you see is brown eyes. A bigtoothed, browneyed person. Now if we assume that the genes that code for teeth or eye color are on different chromosomes, and this is a key assumption, we can say that they assort independently. Let me write that down: independent assortment. So this is a case where if I were look at my chromosomes, let's say this is one homologous pair, maybe we call that homologous pair 1, and let's say I have another homologous pair, and obviously we have 23 of these, but let's say this is homologous pair 2 right here, if the eye color gene is here and here, remember both homologous chromosomes code for the same genes. They might have different versions. Those are alleles. And if teeth are over here, they will assort independently. So after meiosis occurs to produce the gametes, the offspring might get this chromosome or a copy of that chromosome for eye color and might get a copy of this chromosome for teeth size or tooth size. Or it could go the other way. Maybe another offspring gets this one, this chromosome for eye color, and then this chromosome for teeth color and gets the other version of the allele. So because they're on different chromosomes, there's no linkage between if you inherit this one, whether you inherit big teeth, whether you're going to inherit small brown eyes or blue eyes. Now, if they were on the same chromosomee let's say the situation where they are on the same chromosome. So let me pick another trait: hair color. Let's say the gene for hair color is on chromosome 1, so let's say hair color, the gene is there and there. These might be different versions of hair color, different alleles, but the genes are on that same chromosome. In this situation, if someone gets let's say if this is blue eyes here and this is blond hair, then these are going always travel together. You're not going to have these assort independently. And these are called linked traits. Let me highlight that. So these right there, those are linked traits. But for a second, and we'll talk more about linked traits, and especially sexlinked traits in probably the next video or a few videos from now, but let's assume that we're talking about traits that assort independently, and we cross two hybrids. So this is called a dihybrid cross. Very fancy word, but it just gives you an idea of the power of the Punnett square. So let's say both parents are so they're both hybrids, which means that they both have the dominant browneye allele and they have the recessive blueeye allele, and they both have the dominant bigtooth gene and they both have the recessive little tooth gene. So this is the genotype for both parents. Both parents are dihybrid. They're hybrids for both genes, both parents. What are all the different combinations for their children? And I could have done this without dihybrids. I could have made one of them homozygous for one of the traits and a hybrid for the other, and I could have done every different combination, but I'll do the dihybrid, because it leads to a lot of our variety, and you'll often see this in classes. So if I'm talking about the mom, what are the different combinations of genes that the mom can contribute? Well, the mom could contribute the brown so for each of these traits, she can only contribute one of the alleles. So she could contribute this brown right here and then the big yellow T, so this is one combination, or she could contribute the big brown and then the little yellow t, or she can contribute the blueeyed allele and the big T. So these are all the different combinations that she could contribute. And then the final combination is this allele and that allele, so the blue eyes and the small teeth. So that's from mom. And, of course, dad could contribute the same different combinations because dad has the same genotype. Let me write that down. Let me just write it like this so I don't have to keep switching colors. Actually, I want to make them a little closer together because I'm going to run out of space otherwise. Nope. Let me do it like that. OK, brown eyes, so the dad could contribute the big teeth or the little teeth, z along with the browneyed gene, or he could contribute the blueeyed gene, the blueeyed allele in combination with the big teeth or the yellow teeth. Not the yellow teeth, the little teeth. That would be a different gene for yellow teeth or maybe that's an environmental factor. So these are all the different combinations that can occur for their offspring. So let's draw call this maybe a super Punnett square, because we're now dealing with, instead of four combinations, we have 16 combinations. It looks like I ran out of ink right there. It's strange why 16 combinations. Let me write that out. Something's wrong with my tablet. Maybe there's something weird. OK, so there's 16 different combinations, and let's write them all out, and I'll just stay in one maybe neutral color so I don't have to keep switching. I could get this combination, so this brown eyes from my mom, brown eyes from my dad allele, so its brownbrown, and then big teeth from both. I could have this combination, so I have capital B and a capital B. And then I have a capital T and a lowercase t. And then let's just keep moving forward. So I could get a capital B and a lowercase B with a capital T and a capital T, a big B, lowercase B, capital T lowercase t. And I'm just going to go through these superfast because it's going to take forever, so capital B from here, capital B from there; capital T, lowercase t from here; capital B from each and then lowercase t from each. You have a capital B and then a lowercase b from that one, and then a capital T from the mom, lowercase t from the dad. Hopefully, you're not getting too tired here. And so then you have the capital B from your dad and then lowercase b from your mom. Two lowercase t's actually let me just pause and fill these in because I don't want to waste your time. There I have saved you some time and I've filled in every combination similar to what happens on many cooking shows. But now that I've filled in all the different combinations, we can talk a little bit about the different phenotypes that might be expressed from this dihybrid cross. For example, how many of these are going to exhibit brown eyes and big teeth? So big teeth, browneyed kids. Let me write this down here. So if I want big teeth and brown eyes. All of a sudden, my pen doesn't brown eyes. So how many are there? Big teeth and brown eyes. So they're both dominant, so if you have either a capital B or a capital T in any of them, you're going to have big teeth and brown eyes, so this is big teeth and brown eyes. Big teeth right here, brown eyes there. Or maybe I should just say brown eyes and big teeth because that's the order that I wrote it right here. Brown eyes and big teeth, brown eyes and big teeth. Even though I have a recessive trait here, the brown eyes dominate. I had a small teeth here, but the big teeth dominate. This is brown eyes and big teeth. This is brown eyes and big teeth. Let's see, this is brown eyes and big teeth, brown eyes and big teeth, and let me see, is that all of them? Well, no. This is brown eyes and little teeth. This is brown eyes and big teeth right there, and this is also brown eyes and big teeth. They're heterozygous for each trait, but both brown eyes and big teeth are dominant, so these are all phenotypes of brown eyes and big teeth. So how many of those do we have? We have one, two, three, four, five, six, seven, eight, nine of those. So we have nine. Nine brown eyes and big teeth. Now, how many do we have of big teeth? Let me write in a different color, so let me write brown eyes and little teeth. Something on my pen tablet doesn't work quite right over there. So brown eyes and little teeth. So let's see, this is brown eyes and little teeth right there. This is brown eyes and little teeth right there. This is brown eyes and little teeth right there. So there's three combinations of brown eyes and little teeth. And if I were to say blue eyes, blue and big teeth, what are the combinations there? Well, this is blue eyes and big teeth, blue eyes and big teeth, blue eyes and big teeth, so there's three combinations there. And if I want to be recessive on both traits, so if I want let me do this. I want blue eyes, blue and little teeth. There's only one. Out of the 16, there's only one situation where I inherit the recessive trait from both parents for both traits. So if you look at this, and you say, hey, what's the probability there's only one of that what's the probability of having a big teeth, browneyed child? And these are all the phenotypes. There were 16 different possibilities here, right? There are 16 squares here, and 9 of them describe the phenotype of big teeth and brown eyes, so there's a 9/16 chance. So it's 9 out of 16 chance of having a big teeth, browneyed child. What's the probability of a blueeyed child with little teeth? 1 in 16. So hopefully, in this video, you've appreciated the power of the Punnett square, that it's a useful way to explore every different combination of all the genes, and it doesn't have to be only one trait. It can be in this case where you're doing two traits that show dominance, but they assort independently because they're on different chromosomes. You could use it where'd I do it over here? You could use it to explore incomplete dominance when there's blending, where red and white made pink genes, or you can even use it when there's codominance and when you have multiple alleles, where it's not just two different versions of the genes, there's actually three different versions. So hopefully, you've enjoyed that. |
63 | Biomolecules | Mendelian Genetics | Hardy-Weinberg equation | Now that we're familiar with the idea of allele frequency, let's build on that to develop the Hardy, do this in a new color, and actually, let me do it right over here, the Hardy Weinberg principle, which is a really useful principle for thinking through what allele frequencies might be, or what probability you would have if you found someone, what percentage of the population might be homozygous recessive, or homozygous dominant, or might be a heterozygote. And it really builds on the work we've already seen with allele frequencies. Now before we go into that, we're gonna make some assumptions, and these are all just assumptions that get us a stable allele frequency in the population from generation to generation. We're going to assume that there's no selection, no natural selection is, or even unnatural selection, is going on that would change the allele frequencies. So it's not like people with one of the alleles or another are going to be more or less likely to reproduce and have viable offspring. We're also going to assume no mutation, so we're going to assume that one of these alleles can't, isn't from generation to generation turning into another one, or turning into maybe a different, a new type of trait, whether it's green eyes, or whatever else it might be. And we're also going to assume large populations. So that would definitely throw out the example that we looked at in the last video, which I did just to understand the notion of allele frequencies, where we said, hey, look, one out of the four of the genes in this population, or onefourth of the alleles in this population, are the dominant brown, while threefourths, or 75 percent, were the recessive blue. We're gonna assume large populations, so many, many, many, and so that's so that if you have very small populations, you can imagine that, depending on how these reproduce, it's very easy to get to changes in frequencies, but at larger populations, that helps us make the assumption that we have stable allele frequencies. So once again, this is also that we have stable allele frequencies. Now based on that, we've already seen if we take the frequency of the dominant trait, which we can denote with p, and to that we add the frequency of the recessive trait, of the recessive, I should say, allele, let me be very careful here, the frequency of the dominant allele, and to that we add the frequency of the recessive allele, what's that going to be? Well, you see in this case, it adds up to 100 percent, or one, and it's always going to add up to 100 percent, or one. Because we're assuming that there's only one of two alleles in the population, so you have 100 percent chance of getting one of these two, that whatever percentage is going to be, whatever the frequency here is, 100 percent minus that is going to be whatever q is. So these two things are going to be equal to 100 percent, or equal to one. And now we can start to do a little bit of interesting mathematics. It'll allow us to start thinking about things like homozygotes and heterozygotes, and so to do that, let's square both sides. So let's square both sides of this, a little bit of algebra in biology class. And so when you square the lefthand side, this is just squaring a binomial, you might want to review it if this looks like Latin to you, there's many algebra videos on Khan academy that go into this, this is going to be p squared, plus two times pq, plus q squared, and of course one squared is still going to be equal to one. Now what are each of these terms here? What are each of these terms? Well, let's just think about something. p squared is the same thing as p times p. Well, p is the frequency of your dominant allele. So this is the frequency of your dominant allele, the percentage of the allele population, I guess you could say, the allele frequency, that is dominant, and you're multiplying that times it again. Well, another way to think about p, is this is the probability, if you were randomly to pick one of these four genes, and here I'm using my oversimplified population, of course, the truths that we're about to surface to be true, you're going to have to assume a large population, but in this one right over here, one way to view p is what's the probability if I were to pick a gene at random, what's the probability that it is the variant, or it is the dominant allele, what is the probability that it represents the brown variant? So that's one way to view p, so, the probability of getting a, let's just write it that way, a capital B, a dominant brown allele. So what's p times p? That's the probability of getting two dominant alleles. Or another way of thinking about it, this is the probability for someone in the population to be homozygous dominant, so it's the probability of someone being capital B and capital B. And so by the same logic, what is q squared? Well, q squared, that's just q times q, q is the probability of getting one recessive allele, so this is the probability of getting two recessive alleles. One from your mother, and one from your father, so this is the probability of, if you were kind of randomly born into this population, of getting two recessive alleles. Now what is this middle term, right over here? Well, p times q, so pq, so one way to think about it, if you said, what's the probability that from your mother, you get, randomly, you know nothing about them, or if you pick a random mother and a random father, what's the probability, I have to be careful here, so if you're just randomly getting alleles, what's the probability that from on one side you're gonna get the dominant, and from the second side you're gonna get the recessive? So that would be pq, that would be p times q, so that's getting it from, say, from one parent, and then that's from the second parent, but what about the other way around? From the first parent, you have a q probability of getting the recessive one, and from the second parent, you have a p probability of getting the dominant one. So there's two ways of becoming a heterozygote. And so if you add these two probabilities, what do you get? These are both pq, I'm just changing the order of multiplication. You sum these two, you get two pq. So this is the probability of being a heterozygote. So this is a pretty neat result. Just by making a few assumptions, and reasoning through this notion of allele frequency, we're able to come up with this expression that actually is fairly powerful in thinking about allele frequency in a population, and actually, the different genotype frequencies in a population. You see it all makes sense, these all add up to one. The probability of someone being homozygous dominant, plus the probability of someone being a heterozygote, plus the probability of someone being a homozygous recessive, they're gonna add up to 100 percent, because someone's going to have to be one of these three things. Now I'm gonna leave you there in this video, and the next video, we're actually gonna use this Hardy Weinberg equation to actually come up with some very interesting results about a population. |
64 | Biomolecules | Mendelian Genetics | Applying the Hardy-Weinberg equation | Let's stick with this idea, the simplification, that there's a gene for eye color, and it only comes with two variants. It has the dominant variant, which codes for brown eye color, and it has the recessive variant, which codes for blue eye color. So if either one of your alleles is this capital B, you're going to have brown eyes, the only way to have blue eyes is to have a lower case, is to be homozygous for the recessive allele. Now let's say that in a population, it's a large population, one that meets all the HardyWeinberg Equilibrium assumptions, let's say that you were to observe that nine percent of this population has blue eyes. So now we're talking about the phenotype. You can actually observe that they have blue eyes. Based on this, can we figure out p, which is the frequency of the dominant allele. Can we figure this out? And can we figure out q, which is the frequency of the recessive allele, can we figure that out as well? I would encourage you to pause this video and based on what we saw of the HardyWeinberg Equation, can we figure these things out, given this information? Well let's revisit the HardyWeinberg equation. We've worked it out in a previous video, but I'll rewrite it right now. It says, the allele frequency for the dominant allele frequency squared, plus two times the dominant allele frequency times the recessive allele frequency, plus the recessive allele frequency squared, is equal to one. And we saw that this just comes from the idea that p plus q is going to be equal to one. There is a 100 percent chance, if you were to randomly pick a gene, that it's one of these two variants. Now when we say nine percent has blue eyes, what does that mean? Well the only way to have blue eyes is if your genotype is homozygous recessive. Because if you have a capital B in here then you're going to have brown eyes. So we can say that nine percent also has this genotype. Or you can say that the frequency in the population of this genotype is nine percent. But we've already seen, that's exactly what this term right over here is. That's this q squared term. This is the probability, one way to think about it, of getting, q of course is the frequency of the recessive allele, now you could view this as the probability of getting two of the recessive alleles. In your population, it's going to be nine percent. So we could say q squared is equal to nine percent. Or another way to think about it, this term over here is nine percent, or 0.09. Nine percent has this genotype, that's what this tells us right over here. So then we can solve for q. If q squared, I'll write it as a decimal, 0.09, that means that q is going to be the square root of 0.09, which is equal to 0.3. Just like that, we were able to figure out the allele frequency of the recessive allele. And I could write that as a percentage, 0.3 or 30 percent, if you were looking at the genes in the population, 30 percent express our code for the recessive allele, or the recessive variant. Based on that, we can figure out what percentage code for the dominant variant. The rest of the genes must code for the dominant one, because we're assuming there's only two of them. P plus q equals 100 percent, or p plus q is equal to one. So this must be 70 percent. So just based on that, we can kind of dig a little bit deeper here. So what is p squared? P squared is going to be 70 percent squared, or 0.7 squared. So this right over here is 0.7 squared, which is 0.49. So one way to think about it is, based on this, and once again, it's a simple equation, but these really neat ideas are starting to pop out of it based on just this information. We're now able to say that 49 percent of the population is going to have a genotype of capital B, they're going to be homozygous dominant. And then we can figure out this right over here. Two times p times q, that's going to be two times 0.7, times 0.3. So let's see, that's going to be two times 0.21, so this right over here is going to be 0.42. Or another way to think about it is, 42 percent of this population is going to have the genotype upper case B and lower case b. And you see they all add up. 49 percent plus 42 percent is 91 percent, plus nine percent all adds up to 100 percent. So you get a little bit of information here, and based on what we know about allele frequencies, making a few assumptions, we're able to get a lot more knowledge about this population. And this is actually very useful in real life, when people think about, say, a recessive allele that might cause some type of a disease, based on the incidents of that disease, people can start to think about, "What percentage of the population is a carrier?" Say they're heterzygotes for that disease. So this is actually very useful in real life. |
65 | Biomolecules | Dna Technology | Gel electrophoresis | Let's say that you have some vials here, and you know that in the solution you have fragments of DNA in each of these, and what you're curious about, well, what about the DNA fragments in our, in this first vial? In vial number one. How long are those fragments? How many base pairs? How long are they? Well, you might say, well why don't I just take them out and count them? Except for the fact that they're incredibly small and incredibly hard to handle. Even a fairly large fragment of DNA, let's say we're talking about something that's on the order of 5000 base pairs, well that's going to be approximately one to two micrometers long if you were to completely stretch it out. And we can't even start to think about how thin the actual diameter is, if we just, but lengthwise, the long way, it's only going to be one to two micrometers which is super duper small. This is one to two thousandths of a millimeter. So that's not going to help us to somehow try to manipulate it physically with our hands or with, you know, kind of rough tools. So how do we do that? And we could have other vials there. How do we see how long the DNA strands that are sitting in those vials actually are? And the technique we're going to use, gel electrophoresis, it actually could be used for DNA strands, it could be used for RNA, if could also be used for proteins, any of these macromolecules, to see how long are those fragments? And so let me write this down. Gel electrophoresis. And it's called gel electrophoresis because it involves a gel, it involves electric charge, and phoresis is just referring to the fact that we are going to cause the DNA fragments to migrate through a gel because of the charge. So phoresis is referring to the migration, or the movement of the actual DNA. So how do we do this? Well here is our set up, right over here. We have our gel, that's inside of a, that's embedded in a buffer solution. So this gel, the most typical one is agarose gel, that's a polysaccharide that we get from seaweed, and it's literally a gel. It's a gelatinous material. And what we're going to do is, is we're going to put, we're gonna take samples, so we might take a little sample from this one right over here, and we'll put it in this well, right over here. And you can view these wells as little divets in the gel. You could take a little sample from here and put it into this well. And then you could put a sample from here, and you could put it in that well. And it's going to be bathed inside of this buffer, so you can see the buffer I drew, this fluid, and that's really just water with some salt in it. And the buffer is going to keep the pH from going too far out of bounds as we place a charge across this entire thing, because if the pH gets too far in the basic or acidic side, it might actually affect the DNA, or affect the charge on the DNA. Now what we're going to do is, we're gonna put a charge across this whole setup. Where the side where the wells are, where we're gonna place the DNA, that's going to be where we're gonna put the negative electrode, so that's our negative electrode there. And the other end is going to be our positive electrode. And we're going to use the fact that DNA has a negative charge at the typical pHs, or the pHs that we are going to be dealing with. Now we can go back into previous videos, and we can see it right over here, you see these negative charges on our phosphate backbone. And so what is going to happen? What is going to happen once we connect both of these to a power source, and then this side is negative and this side is positive? Well the DNA is going to want to migrate. Now, let's think about what will happen. Will shorter things migrate further, or will longer things migrate further? Well you might say, well longer things are going to have more negative charge, so maybe they go farther away, but then you also have to remember that they're also moving more mass. So their charge per mass is gonna be the same regardless of length. And so what determines how far something gets, how much it migrates over a certain amount of time, is how small it is. Remember, we have this agarose gel, and people are still studying the exact mechanism of how this DNA, or these macromolecules, actually migrate through the polysaccharide, but if you imagine this polysaccharide is kind of this mesh, this net, this sieve, well smaller things are gonna be able to go through the gaps easier than the larger things. And so if you let some time pass, if you let some time pass, some of the DNA, let's say this DNA, gets around there. Let's say, and I'm just color, you actually wouldn't see these colors, let's say this DNA gets around that far, so it doesn't get as far. Let's say that this DNA doesn't migrate, let's say it has some that migrates that far and let's say it has some that migrates that far. And so if you just saw this, you wait some amount of time, and you were come back and you were to see this migration, you were to see this migration occur, and the longer you wait, the further these things are gonna get. In fact, if you wait too long they're gonna fall off all the way over the other edge. Is, if you just saw this you'd say okay, well this strand right over here these must be smaller DNA molecules. They must be shorter. These must be a little bit longer, and these must be even longer than that. And this grouping right over here is going to be the longest of all. So this was a mixture of some longer strands and still longer ones, but not quite as long. And, for example, maybe there are some really short strands, maybe there were some really short strands in that, what I'm drawing as, that orange group right over here. So, what I just did right over here this could tell you the relative length of these strands but how would you actually measure them? Well that's where you can go find standardized solutions, which we call a DNA ladder. And so let's say you go get the DNA ladder, I'm gonna draw it in pink, so you literally could buy this. You can buy it online. And the standard solution let's say it separates like this. So it separates, that goes there, let's say some of it goes like, there, and some of it goes like, there. Well you would be able to know from the labeling, or whichever one you choose to buy, that this grouping here, this all of the DNA that is 5000 base pairs let's say. Let's say this right over here is 1500 base pairs. And let's say this over here is, let's say this over here is 500 base pairs long. And so now you can use this DNA ladder, these standardized ones, to gauge how long, how many base pairs these are. So you say okay, this blue one here, this is a bunch of DNA that's a little bit longer than 500 base pairs but it's shorter than 1500 base pairs. You can see this green one here, well it's a little bit longer than 1500 base pairs, it didn't migrate quite as fair as this big bundle of 1500 base pairs guy did. And so then you can get a better approximation. And you can choose your ladder based on what you think you are going to find there, what you're actually going to look for. Now the other thing to appreciate is, when you see, when you see the DNA having migrated this far, you might say okay, is this one DNA strand, is that one DNA strand that I'm looking at? And just going back to the measurements, no. That is many, many, many, many DNAs that you're looking at. And this is, they're not all stretched out like that. Remember, even something that is 5000 base pairs long is only going to be one to two micrometers if you stretch it out. So, you wouldn't even be able to see it, it's a thousandth of a millimeter. You wouldn't you even be able to see it. So this is many, many, many molecules of DNA, is migrating that far. And they wouldn't even have to be that small to be able to migrate through that polysaccharide gel. Now the last thing you're probably saying is okay, wait, but how am I even seeing it over here? How do I actually see this DNA? Especially if they're these super, super small molecules? And the answer is you put some type of marker on the DNA, that will make them visible. Some type of dye, or something that might become fluorescent. And one of the typical things that people often use it ethidium bromide. And ethidium bromide is called an intercalating agent, and it's a molecule, you can see the ethidium right over here, these are two DNA, two backbones of DNA, you can see the base pairs bonding here, and then this right over here that is ethidium that has fit itself, that's why we call it intercalating, it has fit itself in between the rungs of the ladder. And when it does so, inside of DNA, it actually becomes fluorescent when you apply UV light to it. So if you put this ethidium bromide into all of your DNA right over here, and then as it migrates, and then if you were to turn on a UV light, it would become fluorescent, and you would actually see these things. And so if you wanted to see what it actually would look like in real life, well this is what it would look like when you were to, if you were to look at it straight on. Where this would have been a well, let me make it a little bit easier to read. So right over here would have been the well, where you would put the DNA ladder, and it would come up with standardized measurements. Maybe that's our 5000 base pairs, this right over here is our 1500 base pairs, and this right over here is our 500 base pairs. And then let's say you had some solution of some other DNA, and you wait a little while, and you see look, it migrated not quite as far as a 500 base pair, so it must be little bit, this must be a bundle of things a little bit longer than 500 base pairs, but for sure a lot shorter than 1500 base pairs. Now once again, doesn't have to have just one fragment length, you could have had another group that was, maybe right at 1500 base pairs. And you've probably seen this, whenever you see people talking about genetic analysis, and things like this, you're often seeing people look at one of these readouts from gel electrophoresis. So now you know what's actually going on here. This isn't a strand of DNA, this is a big, this is a bunch of DNA that has been tagged with some type of a dye, or the ethidium bromide, or something like that. And it's a bunch of those molecules and they've migrated based on the charge. They're trying to get away from that negative charge to the positive charge. And the smaller molecules, this is a bunch of small molecules, right over here, are able to get further because they're able to get through the mesh of the agarose gel. |
66 | Biomolecules | Dna Technology | Polymerase chain reaction (PCR) | I'm here with Emily, our biology content fellow, to talk about PCR, or Polymerase Chain Reaction, which you've actually done a lot of. Why have you done PCR? PCR was kind of the mainstay of my graduate project, where I built all sorts of different recombinant DNA molecules, and used them to learn things about plants. And so what does PCR in particular do? PCR basically makes you a lot of copies of a particular fragment of DNA that you're interested in. And so how does that... Why would you need to make a lot of copies of a particular fragment of DNA? So you might want to be making lots of copies so that you can clone it into a plasmid, and then do some other experiments with it, that's a big use. So when we talked about cloning, and we're talking about sticking a fragment of DNA inside of a plasmid, it's not like you're just sticking one fragment into one plasmid, you're doing that with many, so you need a lot of fragments of DNA. Exactly. That is exactly it. And you might start with a very small sample of DNA. Where else would you have to do PCR? PCR is used a lot in forensics, it's also used a lot in medical diagnostics, so this could actually be your DNA that was being checked to see if you have a gene that would predispose you to a particular condition, all sorts of really practical applications. Because it's hard to identify just one fragment of that gene. So you want to make copies, or as they say amplify it, so that you could run it in gels and stuff and see how all of those molecules, how big they are or something like that. Exactly, if you were just looking in your DNA pulled out of your cell, that would be a needle in a haystack. So this is how you can really zoom in and look at just the thing you need to see. Okay. So, you've drawn some diagrams here, and I actually have never done PCR, but you have, so I'm going to tell you how I understand it happening, and then you tell me if this makes sense. So, what you drew over here, this is doublestranded DNA, and this could have been from a sample of someone's hair, or whatever else, and let's say we want to replicate or make many, many copies of a fragment of this. So let's say the fragment that we really care about is the fragment roughly from there, to... There. This part is what we want to make multiple copies of. And so this first step, denaturation... I have trouble pronouncing things. It's a weird word. It's a weird word. You have 96 degrees Celsius, so this is almost at the boiling point. So it's quite hot, and that separates the two strands. Precisely. And so once they're separated, then you can cool things down, although this still isn't that cool. 55 degrees Celsius would be very uncomfortable. But you would cool it down to this, and then these primers show up. And so one thing to remind ourselves is, this process is happening inside of a test tube, or in a big solution, So you heat it up, the DNA, the two strands separate, and do you just have this primer lying around? So the primer is something that you've ordered from a company and you've ordered a lot of it, so you put in a ton of primer in your reaction, so that there's a really good chance that when you get to this step here called annealing, that a primer is going to bind to many of your pieces of DNA. So if this is our solution, is this all happening in water? Water with some salts and stuff floating around, yeah. Okay. So we have our solution right over here. You'd put whatever your initial DNA sample is in there, and once again it's a very small amount, you'd put a lot of that primer, so you'd want to put that in a lot of surplus, so let me do that in this magenta color. You obviously wouldn't see it in real life, it would just all dissolve. It would just look like a drop of liquid. It would look like... But for visualization, you'd put a lot of primer, and so you heat it up, the DNA strands separate, and then when you cool it back down, this primer is going to be specific to the ends of the region that you want to copy. Exactly. And so when you order online or wherever that you want a certain primer, you're going to pick the sequence of that primer to be specific to the regions you want to copy. Exactly. That's super important. Okay. And so when you cool it back down, the primer attaches, and then you heat it back up, not quite to the 96 degrees Celsius, but to the 72 degrees Celsius, where you extend those. And I'm assuming since it's called Polymerase Chain Reaction, that this is where the polymerase is involved. That is exactly where the polymerase comes in. So the polymerase is what is actually extending this. So I'll just draw a polymerase enzyme right over there, doing the extending, and is it any type of polymerase enzyme? Can I just take the polymerase from my cells and throw it in there? So you actually need a special polymerase, because you need one that is going to be pretty heatresistant, so as you were mentioning, even the cool step of this process is not something that your body would want to be hanging out in. So the polymerase is actually from a really heattolerant microorganism. And what is that? It's called a taq polymerase? It's thermophilus aquaticus, I think? Makes quite a mouthful. And they found it at heated vents, this organism that is able to stand these high temperatures. But that I guess leads to another question, which is why do you have to heat it up to begin with? I guess just to separate the two strands? That's really the key reason. You just have to get them apart, you don't have an enzyme to do it the way you might in a cell, so heat does the trick. Okay, so I get it. So this is one step, I'm getting at least the polymerase part of the PCR, where you heat it up, the strands separate, then you have all of this extra primer there. Because there's so much primer, the primer is much more likely to bind to, at least at this part of the sequence, then for these two strands to get back together at this point, and then you have the polymerase, the taq polymerase in particular, and you would have added that at the beginning, the taq polymerase, I guess I'll put it in a yellow color. So you would also put all that taq polymerase in there. And once again, these things aren't robots, they don't know exactly what they need to do, they just bump into things in the right way and react in the right way, and then you would also have to add a bunch of nucleotides. Yes, absolutely. Your reaction is not going to work if you forget the nucleotides. So, the taq polymerase, when you heat it back up again after the primers have been attached, is going to start adding all of these nucleotides. And what, do you just wait a certain amount of time? Will it just keep going on forever? It'll keep going on for a while, usually you do pick the length of that step to match how much time you expect the polymerase to need to complete your fragment. But it kind of will stop. Either it will fall off or it'll stop when you go on to the next step. Okay, so this, I get this so far. So, so far we have after one cycle, what you've written here, after one cycle we would have doubled at least that part of the sequence that we care about. Although we might have copied even beyond that sequence. So, where does the chain reaction come into this? So I guess you can interpret chain reaction in two ways, and one is that's sort of what the polymerase does, is you know, add things to make a chain, but there's actually even more of a chain reaction to mention here, and that's that we're actually getting this kind of exponential process going on. So you do it one cycle, you get to this situation right here, you heat it up, the strands separate, you cool it down, the primers attach, you heat it up again, the taq polymerase does its job, and like all polymerase, it goes from the 5 prime to the 3 prime direction, we talked about that in the application. Precisely. So now you have two strands, but now, since all of that stuff is in that solution, you can heat it up again, now these two strands can turn into... Or these two double strands can now turn into four single strands, then you can cool it down again. Now, they get primers attached to them, and they're still the same primer, because we still care about the same sequence. And so now you go from one to two to four, and so you keep repeating this. And so how many times would it be typical for you to repeat this cycle? So like, 35 might be a pretty typical number of cycles to do. It depends a little what you're doing. But you're going to do it a lot of times. And so if you do this 35 times, I mean each time you're multiplying by 2. So it would be 2 to the 35th power, which is well over a billion times, so how long would that take? You've done this before. It depends on the length of your fragment, but usually like two to three hours. So in two to three hours, you can start with one fragment, and get into the billions. If it's perfectly efficient, which I wish it always were, but you usually get quite a few pieces made. And one thing that I've always wondered when I first learned about this, and I'd like to go into a lab and do this with you, is okay, I get that you have your primer, and then the polymerase is just going to extend it, like that, but I was like well, it's going to be... How does it know where to stop? And you explained, well, on that first pass, it might not know where to stop, but then when you start going in the other direction, it's going to, so over here, when it goes in the other direction, it's going to hit a... It's not going to have anything else to copy. Exactly. So most of the billions of molecules that you produce, are going to be both ends kind of a nice clean cut. The vast, vast majority, exactly. Fascinating. |
67 | Biomolecules | Dna Technology | DNA libraries & generating cDNA | Alright, so let's say that you've got this little guy over here and he's got his shoes and he's just happy, smilin'. So, this guy right here is our protein. So, let's look at how this protein was created. So, in order to make protein we have to start out with our base, and in this case our base is DNA. >From DNA we generate messenger RNA, then that messenger RNA eventually leads to the formation of a protein. And protein is this happy guy over here. This is pretty straightforward, but what if we wanted to go in reverse? What if we started out with a protein and we wanted to figure out what its DNA sequence was? So, if we wanted to go in this direction. So let's look at how this is done. Now scientists thought it would be nice to basically be able to type in the name of any protein that they're interested in and automatically it would pop up with the DNA sequence of that protein. Now that is known as a DNA library. And a DNA library would be beneficial for researchers, and scientists, and clinicians. So, let's look at how this is done. So, we'll start out with our protein and our protein is basically a chain of amino acids. So, amino acids basically are formed from messenger RNA. So, if we know the amino acid sequence of our protein we know what the messenger RNA sequence is based on the Codon table, that we all are too familiar with. So, if we have the messenger RNA sequence, what we do is we add an enzyme known reverse transcriptase and when we add reverse transcriptase, basically takes this messenger RNA and makes a complimentary DNA sequence to the messenger RNA. And that's known as cDNA, the 'c' stands for complimentary. So complimentary DNA, one thing to keep in mind is singlestranded DNA. So, normally DNA in our cells is doublestranded DNA, but complimentary DNA is singlestranded. So, in order to generate doublestranded DNA we need to add another enzyme known as DNA polymerase. DNA polyermase basically generates doublestranded DNA. So, this is basically step one of the process of creating a DNA library. So this is step one, now let's look at step two. So, now that we have our doublestranded DNA what we need to do is sequence it. So, in order to sequence it we'll start out with our doublestranded DNA and we'll basically inject it into some sort of cloning vector, such as a plasmate or a virus. Cloning vector, and that cloning vector can then be, then you can take that cloning vector and add it to some bacteria. And it'll basically infect the bacteria and the bacteria will basically produce lots and lots of this DNA, this doublestranded DNA, so that's a process known as Amplification. And once we have lots of doublestranded DNA, we'll go and sequence that doublestranded DNA and basically once we have that sequence we'll put the sequence into a large database that's readily accessible online and that database will basically populate the DNA library. So, now anybody that is interested in the DNA sequence of a particular protein can just go into this library, and pull up the genetic sequence of the protein of interest. |
68 | Biomolecules | Dna Technology | DNA cloning and recombinant DNA | Let's talk a little bit about DNA Cloning. Which is all about making identical copies of a piece of DNA. And usually it's a piece of DNA that codes for something we care about, it is a gene that will express itself as a protein that we think is useful in some way. Now you might have also heard the term cloning in terms of the Clone Wars in Star Wars or Dolly the sheep and that is a related idea. If you're cloning an animal or an organism, like a sheep, well then you are creating an animal that has the exact genetic material as the original animal. But when we talk about cloning and DNA cloning we're talking about something a little bit simpler. Although, as we'll see, it's still quite fascinating. It's identical copies of a piece of DNA. So how do we do that? Well let's say that this is a strand of DNA right over here and I'm just drawing it as a long, but this is a doublestranded, and I'll just write it down, this is double stranded. I don't want to have to take the trouble of keep drawing the multiple strands. Actually, let me just draw, let me just try to draw the two strands just so we remind ourselves. So there we go. This is the doublestranded DNA and let's say that this part of this DNA has a gene that we want to clone. We wanna make copies of this right over here. So gene to clone. Gene to clone. Well, the first thing we wanna do is we wanna cut this gene out some how. And the way we do that is using restriction enzymes. And there's a bunch of restriction enzymes, and I personally find it fascinating that we as a civilization have gotten to the point that we can find and identify these enzymes and we know at what points of DNA that they can cut. They recognize specific sequences and then we can figure out well which restriction enzyme should we use to cut out different pieces of DNA, but we have gotten to that point as a civilization. So we use restriction enzymes. We might use one restriction enzyme, Let me use a different color here, that latches on right over here and identifies the genetic sequence right over here and cuts right in the right place. So that might be a restriction enzyme right over there and then you might use another restriction enzyme that identifies with the sequence at the other side that we wanna cut. So let me label these. These, those things right over there those are restriction enzymes. Restriction enzymes. And so now you would have, after you applied the restriction enzymes, you will have just that gene. You might have a little bit left over on either side but essentially you have cut out the gene. You've used the restriction enzymes to cut out your gene and then what you wanna do is you wanna paste it into what we'll call a plasmid. And a plasmid is a piece of genetic material that sits outside of chromosomes but it can reproduce along, or I guess we can say can replicate along with the machinery of the, the genetic machinery of the organism. Or it can even express itself just like the genes of the organism that are in the chromosomes, express themselves. So then so this is where we cut, let me write this, we cut out the gene and then we wanna paste it then we wanna paste it into a plasmid. And plasmids tend to be circular DNA so we will paste it into a plasmid. And in order for them to fit there's oftentimes these overhangs over here. So you might have an overhang over there, you might have an overhang over there. And so the plasmid that we're placing in might have complimentary base pairs over the overhangs, which will allow it easier, it will become easier for them to react with each other if they have these overhangs. So let me, we're pasting it into the plasmid. And this is amazing because obviously DNA, this isn't stuff that we can, you know, manipulate with our hands the way that we would copy and paste things with tape. You're making these solutions and you're applying the restriction enzymes. The restriction enzymes are just in mass cutting these things. They're bumping in just the right way to cause this reaction to happen then you're taking those genes and you're putting them with the plasmids that happen to have the right sequences at their ends so that they match up and then you also put in a bunch of DNA ligase. DNA ligase, to connect the backbones right over here. And we also saw DNA ligase when we studied replication. So that is DNA ligase, which you can think of it as helping to do, helping to do the pasting. And so now we have this plasmid and we want to insert it into an organism that can make the copies for us. And an organism that's typically used, or a type of organism is bacteria and E. coli in particular, and so what we could do is, we could, let's say that we have a bunch, let's say you have a vial right over here. You have a vial and it has a solution in it with a bunch of E. Coli. A bunch of E. coli. And you actually wouldn't be able to see it visually but there is E. coli in that solution. And then you would put your plasmids, which would be even harder to see, in that solution and somehow we want the E. coli, we want the bacteria to take up the plasmid. And the technique that's typically done is giving some type of a shock to the system that makes the bacteria take up the plasmids. And the typical shock is a heat shock. And this isn't fully understood how the heat shock works but it does and so people have been using this for some time. So if you have a bacteria, you have a bacteria right over here, it has its existing DNA, so this is its existing genetic material right over there, let me label this. This is the bacteria. You put it in the presence of our plasmids so you put it in the presence of our plasmid and you apply the heat shock and some of that bacteria is going to take in the plasmid. It's going to take in the plasmid. And so just like that, it's going to take it, it's going to take it in. And so what you then do is you place the solution that has your bacteria, some of which will have taken up the plasmid, and you put it and then you try to grow the bacteria on a plate. So let me draw that. So let me draw, so here we have a plate to grow our bacteria on, and it has nutrients right over here that bacteria can grow on. It has nutrients. It has nutrients, and so you could say, okay well put this here and then a bunch of bacteria will just grow. So you would see things like this, which would be many, many, many cells of bacteria, there would be colonies of bacteria. You could just let them grow but there's a problem here. Because I mentioned some of the bacteria will take up the plasmids and some won't. And so you won't know, hey when this bacteria, when it keeps replicating it might form one of these, it might form one of these colonies. So this is a colony that you like. So this one is a good colony, put a checkmark there. But maybe this colony is formed by an initial bacteria or a set of bacteria that did not take up the plasmid so it won't contain the actual gene in question. So you don't want that one. So how do you select for the bacteria that actually took up the plasmid? Well, what you do is besides the gene that you care about that you want to make copies of, you also place a gene for antibiotic resistance in your plasmid. So now you have a gene for antibiotic resistance here, and so only the bacteria, and I think it's amazing that we as humanity are able to do these types of things, but now only the bacteria that have taken up the plasmid will have that antibiotic resistance. And so what you do is in your nutrients you grew nutrients plus antibiotics, plus an antibiotic. Antibiotic, and so this one will survive 'cause it has that resistance. It has that gene that allows it to not be susceptible to the antibiotics. But these are not going to survive. They're not even going to happen. They're not even going to grow because there's antibiotics mixed in with those nutrients. And so this is a pretty cool thing. You started with the gene that you cared about, you cut and pasted it into our plasmid. Let me write the labels down, into our plasmid that also contained a gene that gave antibiotic resistance to any bacteria that takes up the plasmid. You put these plasmids in the presence of the bacteria or you provide some type of a shock, maybe a heat shock, so that some of the bacteria takes it up and then the bacteria starts reproducing. And as it reproduces it also is reproducing the plasmids and because it has this antibiotic resistance it is going to grow on this nutrient antibiotic mixture and the other bacteria that did not take up the plasmids are not going to grow. And so just like that you can take this, you can take this colony right over here, and put it into another solution or continue to grow it and you will have multiple copies of that gene that are inside of that bacteria. Now the next question, and I'm over simplifying things fairly dramatically is well you now have a bunch of bacteria that have a bunch of copies of that gene, how do you make use of it? Well, the bacteria themselves, let's say that gene is for something you want to manufacture say insulin for diabetics, well you could actually use that bacteria's machinery, we used its reproductive machinery to keep replicating the genetic information, but you can also use its productive machinery, I guess you could say, it's going to express its existing DNA but it can also express the genes that are on the plasmid. And in fact that's what would give the bacteria its antibiotic resistance but if this gene was say for insulin, well then the bacteria will produce a bunch of insulin, a bunch of insulin molecules, which you might be able to use in some way. And I'm not going to go into all the details of how you will get the insulin out and how you could make use of it, but needless to say, it's pretty cool that we can even get to this point. |
69 | Biomolecules | Dna Technology | Hybridization (microarray) | So in this video, we're going to be talking about something known as DNA hybridization. DNA hybridization. Alright, so in this video we're going to be talking about something known as DNA hybridization. So, DNA hybridization. Now, what is DNA hybridization? Well, basically what it... So, let's work through an example to try and explain what DNA hybridization is. So, let's imagine that we have two cells. So over here we have Cell A and over here we have Cell B. Now, let's imagine that Cell A is a cancer cell. So, this is a cancer cell. And Cell B over here is a normal cell. So, this is normal. Now, cancer cells basically have the ability to proliferate and grow and grow and metastasize and move throughout the body. So, basically they have this unregulated cell growth. And the reason that the cell growth is unregulated is because there are various mutations that cause changes in the proteins that are expressed and changes in the regulation of the cell cycle. And there are hundreds and hundreds of different mutations and hundreds of different proteins that could be effected. And, all of them can lead to cancer. Now, one is producing different proteins in different amounts. Now, what are kind of the two options that we have for certain genes? So, let's imagine that we have Gene A over here. So if this is Gene A, what are the two options? Either Gene A can be upregulated or it can be downregulated. So if it's upregulated then what we have, is we have the gene products, which is mRNA and eventually protein, we have a lot more of the mRNA and the protein that Gene A encodes for. And what that basically means is that, let's imagine that Gene A encodes for a protein that will induce cellular proliferation and will allow that cell to go and metastasize throughout the body. Well, if we have a lot more of Gene A being expressed, either because the promoter is upregulated or for whatever reason, now we have lots and lots of this protein that basically allows cellular proliferation to occur. We have lots of this protein floating around the cell and we have this cancer cell proliferating uncontrollably. So, another option is if we have Gene B, so if we have Gene B. Gene B could be downregulated. And that basically means that Gene B isn't producing its gene product. And what if that gene product were something that basically stopped this cell from proliferating. Well, if we have less inhibition then we basically have more proliferation. And the third option for any specific gene in a cancer cell, so let's say Gene C, is that there's no change. So, there's just no change. So, we what we want to do is use DNA hybridization technology in order to assay the gene transcription profiles of a cancer cell compared to a normal cell. And in order to do that, we need to use something known as a microarray. So, a microarray. Now, what is a microarray? Well, array basically means that we're assaying a whole bunch of different things. And in this case, we're assaying the transcription profiles of a bunch of different genes. And micro just means that it's small. So, this could be as small as a chip. So, let's imagine that we have a microarray chip. So, let's say that we've got this chip and it's basically just this square. And this chip has a lot of different holes in it. So let's imagine that we've got lots and lots of holes. So we have just hundreds of these holes. And I'll just draw a few for simplicity's sake. So we have a bunch of these holes on the mircoarray chip. Now these holes are actually little tiny wells, they're microscopic wells. So if we actually looked at this from the side, so here's the chip, we're looking at it from the top. It's lying on the table, we're looking down at it. If we looked at it from the side, one of these wells would look like this. And inside the well would be the, would be a complimentary mRNA strand. So, we've got just lots and lots of these little complimentary mRNA strands. And what are they complimentary to? Well, they're complimentary to a specific gene. So, let's say that inside one of these wells, let's draw another well over here. Let's say that inside one of these wells, we have the complimentary mRNA to Gene A. So we've got the complimentary mRNA to Gene A. Now let's imagine that in this cancer cell, Gene A is upregulated for whatever reason. And if Gene A is upregulated, it's being overtranscribed and that means that there's lots and lots of the Gene A mRNA floating around in this cell. So there's just a bunch of the Gene A mRNA. And this is in comparison to the normal amount of Gene A products, which might just be a few Gene A mRNAs. Now, what we can do, is we can take this cell and we can break it apart. And we can label the mRNA with a certain color. So let's say that I label each one of these mRNAs with a yellow fluorescent label. So, let's imagine that I labeled every single one of the mRNAs with a yellow fluorescent label. And let's imagine that I labeled the mRNA in the normal cell with a blue fluorescent label. Now what I can do is I can break these cells apart and I can basically add the intracellular contents to this well. So, I can add it to this well. And since I have lots and lots of this mRNA that's labeled yellow, what I'm going to have, I'm going to have a lot of the mRNAs binding to the complementary strands. And so I'm going to have a really bright yellow well. And when I add the normal cell intracellular contents, I'm going to have some blue. So, I'm going to have lots of yellow and a little bit of blue. And what that'll basically look like is, it'll really just, you won't be able to see the blue, it'll really look like just a bright yellow dot. So, let's imagine that this is the well. It'll look like a bright yellow dot. And a computer can scan every single one of these wells and basically decide, "okay, is it a brighter yellow or is it a brighter blue?" If it's a brighter yellow color, if you see mainly yellow, that means that you have a lot more of that specific gene's products being expressed in the cancer cell compared to the normal cell. Now, let's imagine that we look at a downregulated gene. So it we look at a downregulated gene, let's just draw another well. So let's draw a well over here. Now, if we look at a downregulated gene, we've got lots of the Gene B mRNA, the complementary strands, inside this well. And we're going to have very few of the Gene B mRNA in the cancer cell and then a lot more in the normal cell. And once again, we'll label the Gene B mRNA with a yellow fluorescent label. And over here, again, I'm sorry, over there, we're going to label it blue. So, we'll label it with this blue fluorescent label. And once again, we're going to lice the cells and expose the intracellular contents to this well. And what we're going to have, we're going to have very few of the Gene B products binding and we're going to have a lot of the normal cell, of the Gene B byproducts in the normal cell, binding. So, when you look at this well, it's going to pop up as mainly blue. And once again the computer's going to read this and it's going to notice, "oh, well, this well has mainly a blue fluorescent label" which means that the normal cell is expressing a normal amount and there's a lot less of that gene being expressed in the cancer cell. So, this is kind of the idea of a microarray chip and assaying the gene expression profile in a cancer cell versus a normal cell. It's able to tell you whether a specific gene is upregulated or downregulated. And you're also able to see if a specific gene has no change and if there's no change, then instead of seeing either a yellow or a blue dot, you would see something kind of in the middle. So, maybe you'd see a green dot. And that's basically a quick way in order to look at a whole bunch of different genes on a single chip and try and quickly determine which gene is upregulated or downregulated in a cancer cell compared to a normal cell. And this can help you tailor your therapies. So, let's say that you know that this well right here is for a specific protein and you have a drug that's able to target that protein. Well, now, you're able to tailor your therapy for that individual patient using this microarray technology. |
70 | Biomolecules | Dna Technology | Expressing cloned genes | Normally when we think about cloning, we think about cloning in this sense. So let's say that we've got a baby and it's just so cute that we want two of them. So we can go and clone a baby. Well this isn't exactly what we're talking about when we talk about cloning in a scientific sense. Normally what that means, scientifically is let's say that I've got a cell and there's a certain gene, let's call it gene A for now, and I want to clone that gene. So how do I go about cloning gene A? Well, the first step is to isolate the messenger RNA from the gene of interest. So if we have the messenger RNA, what we need to do is we need to convert it into DNA. So if we add reverse transcriptase, which is an enzyme that will go and actually create DNA from RNA we'll end up with something known as complimentary DNA. Now complimentary DNA, and that's what the C stands for, is basically the complimentary DNA sequence to the MRNA sequence. Now keep in mind that the complimentary DNA only contains exons, so the introns have already been spliced out. We talk about introns and exons in another video. So next, what we have to do is we take this CDNA, this complimentary DNA, and now what we want to do is amplify it. We want to create lots and lots of the complimentary DNA so that we can have plenty of CDNA to work with. In order to do that, we have to take this complimentary CDNA and transform it into a plasmid. Now, what does transform mean? What it basically means is that we're taking the complimentary DNA and we're basically putting it into a plasmid. So we're gonna transform it into a plasmid and this plasmid is going to contain antibioticresistant genes. Antibioticresistant genes. We'll talk about why that's important in just a second. Now that we have this plasmid, we want to actually infect bacteria with the plasmid. So we want to put it into bacteria. Once it's in the bacteria, what we want to do is we want to actually add antibiotics to the cultured bacteria. So we're gonna add antibiotics and since the plasmid contained antibioticresistant genes, the bacteria that were successfully transfected will survive and all the other ones that didn't have the plasmid inserted will die. So now we've got pure cultured bacteria that all contain the plasmid which contains the gene of interest, the DNA of interest, and now the bacteria just does its thing so it's just gonna start to replicate. The bacteria is gonna replicate and as it's replicating, it's gonna produce lots and lots of the gene of interest. It's gonna produce lots of the MRNA of interest. Basically what we just did through this process is we just cloned the gene of interest. And that's what we're talking about when we talk about cloning, at least from a scientific point of view. |
71 | Biomolecules | Dna Technology | Southern blot | So in this video, I'm gonna be talking about something known as a Southern Blot. So, a Southern Blot basically allows you to visualize a specific piece of DNA that you're interested in. So let's imagine that we have a cup and it's filled with DNA. So it's got just a whole bunch of DNA inside. And there's just lots and lots of those DNA and let's imagine that I'm specifically interested in one gene. So let's imagine that I'm interested in Gene A and I want to see if Gene A is inside of this cup. If it's inside of this long piece of DNA. Now, in order to figure out whether or not Gene A is inside this cup, basically we have to do this process known as a Southern Blot. And we'll break it up into a couple of different steps. So Step 1, what we're gonna do is we're gonna take this DNA and we're gonna cleave this. So, "take the DNA and cleave it." So, let me draw that out. So, we're gonna take this big old strand. We're gonna remove it outside of the cup over here. So, we got this big strand and we're gonna cut it up. We're gonna expose it to enzymes that will basically cleave the DNA in a whole bunch of different parts. And that will result in lots of these smaller pieces of DNA. So that's basically the first step. So we got a bunch of small little pieces of DNA. Now Step 2, what do we do? Well, what we're gonna do is we're gonna take all these tiny little DNA fragments and we're gonna run them on the gel. So, specifically we're gonna do a gel electrophoresis, "electrophoresis" on these DNA fragments. And I made a video on gel electrophoresis if you want to refresh, you can watch that video. But basically, the gel electrophoresis will help us separate these DNA fragments based on size and based on charge. So, let's just diagram that out. So, we're gonna take these DNA fragments and we're gonna run them on a gel. So, let's imagine that this is the gel and we add the DNA fragments to different wells. So the fragments are gonna move down the gel and they're gonna basically be separated based on size and based on charge. So, we're gonna have these fragments separated like so. So now, we've got this gel and we've got the DNA fragments separated by size on this gel. So the next step, step number three is basically we're gonna take this gel and we're gonna transfer it to a filter. So, transfer the gel onto a filter. And what the filter will basically allow us to do is it allow us to visualize 'cause this gel is very flimsy. So, we want to transfer it onto a filter. What we'll do is we'll take a filter that's basically the same size as the gel and we're gonna basically just put it right on top of the gel for a little bit and the fragments will basically transfer on to the filter. So now, we're gonna have a filter with these fragments and the filter is a lot sturdier than the gel. So this is the filter and I'll just write that down over here and this over here is the gel. Okay, so the next step, step number four that we're gonna take the filter and we're gonna expose it to a radiolabeled the piece of DNA. So, "expose to radiolabeled DNA." Now, this radiolabeled DNA is going to be the complement to our gene of interest. So, we're interested in finding out if Gene A is present in this mass of DNA over here. So what we do is we're gonna take the complementary sequence to Gene A and radiolabel it and expose it to this filter. So, let's imagine that the radiolabeled piece of DNA is this pink piece of DNA. And let's imagine that we do have Gene A, so let's imagine that this piece of this DNA fragment was actually Gene A or our gene of interest. So what's gonna happen is when we expose the radiolabeled DNA to this filter paper, it's going to anneal to our gene of interest. So we're gonna have this radiolabeled piece of DNA stuffed to this DNA fragment which it's complement. So, in order to visualize it, in order to visualize this radiolabeled piece of DNA, we have to do the fifth and final step which is expose the filter to an xray film in order to visualize the radiolabeled probe. So, "expose to xray." And the xray basically it will shoot a bunch of xrays and since this piece of DNA is radiolabeled, it will pop up on the xray film. So, we're gonna have a film and we'll draw that film over here so we'll have this film and basically the only thing that will pop up is this fragment over here and that fragment will have a control and we'll be able to say, "Okay. Well, since we have this fragment "it's basically the radiolabeled piece" "of DNA and since we see the radiolabeled DNA" "it means that it had bound." "It was bound to this Gene A" "which means that Gene A was in this cup of DNA." |
72 | Biomolecules | Dna Technology | DNA sequencing | Have you ever wondered how we sequence DNA? Well, let's just take a quick look at DNA sequencing. We're going to break down DNA sequencing into three different steps. The first step is you take the sample of DNA that you are interested in sequencing and you basically use PCR to amplify the sample. By using PCR in order to amplify the sample, you're able to generate lots and lots of DNA fragments. The next thing that you do is normally in PCR you have to add nucleotides, you have to give the growing strand the substrate from which it can grow. Normally you add in regular deoxynucleotides and those look something like this. You've got an OH group here. You've got an H group here. You have a base... And then you've got a carbon group... And oxygenhydrogen. So, this is what a normal nucleotide looks like... But interspersed in the PCR, what you also want to add is you want to add in something known as a dideoxynucleotide. A dideoxynucleotide looks something like this. It's basically exactly the same thing but it only has a hydrogen here, so this oxygen is removed. And what that basically does is if this dideoxynucleotide, we can abbreviate ddNTP, if this incorporates into the growing strand, since there's no oxygen group here, the strand can no longer elongate. You basically have termination of strand elongation, as soon as this ddNTP incorporates. What you can do is you can actually fluorescently label the different dideoxynucleotides. For example, we have four different options. We can label all the G's blue, we can label all the A's red, all the T's green, and all the C's orange. And so basically what you have is you have these dideoxynucleotides with different fluorescent labels getting incorporated into the growing strand and since PCR is able to amplify creating millions and million of DNA fragments, you can basically, what you can do is you'll have strands of different lengths. Let's just kind of look at an example. Let's imagine that we've got a nucleotide being incorporated here, a regular nucleotide, and then another one incorporated here and then another one and then just randomly, all of a sudden, we have a dideoxynucleotide being incorporated here and this would stop the elongation of the strand. So, you would have a DNA strand which that's just four nucleotides long. And after another round of PCR, what we might have is we might have, one, two, three, four, five, six, it's just growing, it's growing, it's growing, and all the sudden, whoa, what happened? You got a dideoxynucleotide being incorporated. And so basically, you just do this and after you've got millions of samples, you will eventually be able to have something that looks like this. You'll have maybe just one regular nucleotide and you've got a dideoxynucleotide incorporated, or you might have maybe, let's say, two of them, so you'll have two and then you've got a... Let's use this color suite we've got here. What you can basically do is you can see you have strands and they're elongating and different strands are terminated at different points by a dideoxynucleotide. And so, basically, the next step, is you use gelelectrophoresis... Electrophoresis... In order to separate the strands by size. So, when you run all the different fragments on a gel, it will separate them by size and then you can just have a computer go in and analyze all the fluorescent labels. So if it sees here, that you've got this blue fluorescent light, then it knows that the second nucleotide in the sequence is a G, so it'll say G. And then, it'll look here, it'll say, okay well this is a C. It'll look here, it'll say we have another G and so on and so forth. And basically computer is able to, by reading these fluorescent labels, these fluorescent tags, it's able to give you a DNA sequence. And so this is basically an overview of how DNA sequencing works. |
73 | Biomolecules | Dna Technology | Gene expression and function | So what is gene expression? Well, it's basically the process where a gene is used to synthesize some sort of product. So you go from a gene to a product. And normally this product is a protein, but sometimes you can have nonprotein coding genes. You can create things like ribosomal RNA, actually let's list these out. You can either have a protein, you can have ribosomal RNA, shortened to rRNA, you can have tRNA, tRNA, you can also have something known as small nuclear RNA. So basically you go from a gene to a product. Now, how do we determine what the function of the gene is? How do we determine a specific gene exactly what does it do? Well, let's imagine a scenario where there's a cell and normally it's able to if you give it milk... So lets imagine that we give it a bottle of milk, let me just draw a little bottle of milk, it's not the greatest bottle in the world, but, let's just imagine this is a bottle of milk, so we'll label that milk. So if you give this cell milk and normally it's able to take the milk and digest it and it's able to use the milk for energy. Well, what if we wanted to figure out what gene is responsible for being able to digest milk. Well, one thing that we can do is if we have an idea of what gene it might be we can just knockout that gene. So let's just imagine that there's a gene here and we imagine that this has something to do with the digestion of milk. Well, if we knock it out and then we give milk to the cell and if it's still able to digest the milk then we know that this gene didn't really have much to do with digestion of milk. But if we knock it out and the cell is no longer able to digest the milk, then we know that this gene had something to do with the digestion of milk. So this process is known as a knockout. So basically, you're knocking out a gene and trying to figure out what the function is of the gene. So if you knock out a gene what happens to the organism? So you basically create a knockout mutiny and study its effects. So another thing you can do is something known as reverse genetics, reverse genetics. So here what you do is first you start with a gene, and then you sequence it. You figure out what is the sequence of the gene. And then what you can do is you can look for other gene sequences somewhere else in the genome that share a similar sequence. So you sequence it and then you look for a homologous sequence somewhere else in the genome. And if you know what that homologous sequence does then you have a pretty good idea of what that gene might do. So if you know that there's this homologous sequence somewhere else in the genome and it goes for a specific protein, and you know the function of that protein, then you know that the gene of interest might create a protein that has a similar function. |
74 | Biomolecules | Dna Technology | Applications of DNA technologies | So what are some applications of DNA technology? All right, well let's first look at medicine. So what are some applications of DNA technology in medicine? The two big things where recombinant DNA technology was first used was to create insulin and human growth hormone. So before the advent of recombinant DNA technology, insulin and growth hormone were really, really hard to manufacture. You basically had to isolate it from another human, purify it and then give it to patients. But with recombinant DNA technology you can basically just grow these proteins in E. coli. You can grow them in culture that of an E. coli bacteria. So this really has changed the way that medicine is practiced and it's really helped a whole bunch of people. Vaccines is another application of DNA technology. A while ago vaccines were made by first denaturing the disease and then after the disease has been weakened they would inject it into a human and they would hope that their immune system would be able to put up a fight against the weakened virus. And that way in the future if they were infected with that virus, they would at least have some kind of immune response towards the virus. The problem with this was that the patient would sometimes get the disease because you're injecting a weak virus but sometimes it wasn't weak enough. So with DNA technology, they can actually recreate the outer shell of the virus and inject that. So it's a lot more cost effective and it doesn't have the risk of actually causing the disease in the host. So this is much safer and is cheaper and it produces a better immune response. Some vaccines that we actually use recombinant DNA technology to create include the hep B virus and the herpes virus and malaria. So these are some applications of DNA technology in medicine. Another cool application of DNA technology is in solving crimes, so in forensics. So there are parts of the genome known as noncoding regions of the genome. And these regions can actually help forensic scientists identify specific individuals so they can look at things like short tandem repeats, STRs, and these are basically short sequences of the DNA like two to six base pairs long, and they're normally found in really high amounts. They're just these short repeats that are found in really high amounts and to varying degrees between different individuals. So if they actually sequence these short term repeats they could identify specific individuals given a DNA sample. They can also look at mitochondrial DNA. So mitochondrial DNA is inherited from your mother and it's found in really high amounts within an individual cell. So even if there is very little sample available, forensic scientists can analyze mitochondrial DNA in order to identify a potential suspect. Another technology that is using forensic science is Y chromosome typing, so that's YSTR. And this is looking at short tandem repeats found on the Y chromosome. So DNA technology has helped scientists pick out individuals that have committed various crimes based on DNA samples that they were able to find. Agriculture is another field that has greatly benefited from recombinant DNA technology. For example scientists can now create crops that are resistant to insects and that are resistant to herbicides and can also delay ripening of the crops so that you can transport the crop from the farm to the store. So by doing this, you're basically able to create more crops to feed a growing population of individuals. And it also helps with the economy because then you've got farmers that are growing all their crops and if there was some sort of bacteria or virus that destroyed their entire crop, then that farmer would not get paid for that season so by transgenically modifying the crop so that it's resistant to specific things, then they're able to grow their crops, sell it, and feed individuals. |
75 | Biomolecules | Dna Technology | Safety and ethics of DNA technologies | So, DNA technologies are really cool and they've provided us with a lot of really good things. However, there are some safety and ethical issues that surround DNA technology. So, back in 1975 there was a conference on recombinant DNA and they concluded that recombinant DNA used for research purposes can be particularly risky. And so they implemented a set of guidelines to try and minimize that risk. The NIH later issued formal guidelines for recombinant DNA work and now they're very well regulated and there are lots of laboratory safety procedures to try and regulate the use recombinant DNA in the lab. So, one example of a safety concern would be what if we transferred... cancer genes. So if we took cancer genes and then we put them into a bacterial genome, then that bacteria could infect someone and it could potentially transfect cancer genes into an individual. So, that would be pretty bad. So, that's one example of a safety concern using DNA technology. Another safety concern is how do we protect researchers that are working with recombinant DNA from being effected by the technology. Well there are a lot of safety guidelines in place to try and minimize any exposure risk that researchers have when working with these recombinant DNA technologies. So some ethical issues that come up... include... if we're able to modify the genome, then imagine that there's a pregnant woman and we're able to sequence the baby's genome, and let's say that we notice that there's some kind of defect. What are the ethics surrounding the correction of that defect? Is it ethical to fix a mutation that might cause a cancer, for example? How do we know that fixing the mutation isn't going to cause some other cancer? How do we know what the longterm effects of genetically modifying an infant's genome are? So these are all ethical questions that kinda surround that. And if we kinda drag it out, what if an individual's perfectly normal genetically, is it okay if we put in genes that help, that make them smarter or faster? What are the ethics surrounding that? So those are some ethical issues. So that would be genetic modification. So another ethical concern that has been brought up is we're able to genetically fingerprint individuals. So forensic scientists are able to pinpoint a suspect's DNA. They're able to figure out what individual left a DNA sample at a crime scene for example. So, we're getting better at genetic fingerprinting but what are the ethics around that? What if the government was able to track every single person that opened a certain door based on the DNA that was left behind, or what if someone took a piece of gum that you spit out on the sidewalk and isolated DNA and was able to track it back to you. So there are some problems around privacy issues. So, I'll write that down here "privacy issues". And with the Human Genome Project, how do we prevent genetic information from being used in a discriminatory manor? So for example if we know that someone has the gene for a specific breast cancer then maybe health insurance companies won't insure that individual, or maybe future employers won't want to offer that individual a job because they know oh, this individual is gonna get breast cancer later on, we don't want her working here. So there are these privacy issues that come up with the ability to be able to sequence and modify a genome. |
76 | Biomolecules | Chromosomal Inheritance | Evidence that DNA is genetic material 1 | One criterion that we use to define biological life, is that the organism must have the ability to reproduce. And the parent organism... Must have the ability to pass on to its offspring... Some sort of material that contains the instructions for living, and we call this material genetic material. What is genetic material? Well, today we know that it's DNA, or deoxyribonucleic acid. But this wasn't always so obvious. We're going to go through a couple of earlier experiments that helped prove to us that DNA is genetic material. The first experiment we're going to discuss, was one that was conducted by a scientist by the name of Friedrich Miescher. And Miescher lived from 1844 to 1895. And Miescher worked with cells that he took out of pus, and today we know that these are lymphocytes. And lymphocytes happen to have very large nuclei, so if that's the cell, the nucleus might look something like that. And of course, there's this material inside the nucleus. And because the nuclei are so large, Miescher was able to isolate the material inside the nucleus. And he analyzed this material, and came to the conclusion that it was made up of two components. The first was protein. And the second was this substance that acted like an acid, and so he called it nucleic acid. Nucleic because it came from the nucleus. And so Miescher was the first one to isolate and identify nucleic acid. The next experiment we're going to talk about was one conducted by a scientist who lived at about the same time. His name was Wilhelm Roux. And he lived from 1850... To 1924... And Roux worked with cells that were dividing, and he analyzed these cells, so I drew a cell here, and I drew a couple of the organelles also, let's just label them. We have the endoplasmic reticulum here, we have a couple of mitochondria. And the singular is mitochondrion. And then we have a couple of ribosomes, I'm just going to abbreviate it like that. And I also drew some vesicles. And of course, in the center, we have the nucleus. And we're going to see that this cell is undergoing mitosis right now, so I'm going to draw the chromosomes like that in the center, I'm going to draw four chromosomes. And I'm also going to draw the mitotic spindle. which will help to separate the chromosomes into the two cells that are being formed. And what Roux noticed, was that when the cell divided, so the organelles were not necessarily distributed in a very organized and even fashion, you can see the cell on the left got three mitochondria, the cell on the right only got two, the cell on the left got three ribosomes, the one on the right only got two, but he noticed that the material in the nucleus divided in an extremely organized fashion, so each nucleus got exactly four chromosomes. And so Roux concluded that because the material in the nucleus was divided in a very orderly and even fashion, that that must be the genetic material. And this makes sense, because the genetic material or instructions for living, is the most important thing that the parent cell has to pass on to the daughter cell. If one of the daughter cells didn't get enough mitochondria, well, that's not so terrible, as long as it has the correct instructions for living, it can make more mitochondria. If one of the cells accidentally didn't get a ribosome, well, if it has the right instructions for living, it can make ribosomes. So the conclusion here, was that genetic material is made up of protein... And, or, nucleic acid. This was the conclusion that Friedrich Miescher's, experiment and Roux's experiment helped prove. |
77 | Biomolecules | Chromosomal Inheritance | Evidence that DNA is genetic material 2 | We spoke about the experiments of Friedrich Miescher and Wilhelm Roux and how their experiments helped show that genetic material is made up of either protein or nucleic acid or perhaps both. The next logical question to ask is, "Well, which one is it? "Is it protein? "Is it nucleic acid? "Or perhaps it's both?" This question was answered with the experiment of two scientists, Alfred Hershey and Martha Chase. In their famous HersheyChase experiment, that was published in 1952, they showed that it's nucleic acid that's genetic material, and not protein. Hershey and Chase worked with bacteriophages. A bacteriophage is a virus that specifically infects bacterial cells. It can also be referred to simply as a "phage." What is a bacteriophage? Well, it has nucleic acid, which can be either DNA or RNA. In this video, I'm just gonna refer to all nucleic acid as DNA, but keep in mind that, when I say that, it can also mean RNA, because in some viruses, the nucleic acid is RNA. That nucleic acid, or DNA, is surrounded by a protein coat. How do bacteriophages infect bacterial cells? Well, they get kind of close to the bacterial cell and sort of sit on top of it and inject their DNA into the bacterial cell. Then, their DNA gets integrated into the bacterial cells' DNA, which I'm gonna draw right here. Here's the bacterial cell's DNA. The virus's DNA gets somehow integrated, and now this bacterial cell's going to produce a whole bunch of viruses. This is some background information. Now let's talk about Hershey and Chase's actual experiment. Hershey and Chase took some phages and they put these pages in a medium, which means a broth that has a lot of nutrients so that these phages can now multiply and reproduce and make a lot more of themselves. But they wanted to label the protein code of the new generation of viruses. How were they gonna do that? They made sure that all of the amino acids that were in this broth had in them radioactive sulfur. They were labeled with sulfur35, which is one of the radioactive isotopes of sulfur. In this way, they can keep track of where the protein's going and what's happening with the protein. The reason that they chose sulfur for this part of the experiment is because they wanted to label the protein, in particular, and sulfur is found in amino acids, which means that finding proteins with sulfur is not found in DNA. So this is a good way to make sure that they label the protein but not the DNA. Now, when this phage, or the phages that are put into the medium, reproduce, they're gonna take nutrients from this broth. Among those nutrients are amino acids, and they're going to incorporate those labeled amino acids into their protein coats. So they produced a generation of viruses that have radioactivelylabeled protein coats. Now they allow this generation of viruses to infect a bacterial cell. You can see, they climb on top of the bacterial cell and inject their DNA into the cell. Remember, their DNA gets incorporated into the DNA of the bacterial cell. The bacterial cell will produce a whole bunch of viruses. Now, take note that the original protein coats, of course, remain outside of the cell. Hershey and Chase now wanted to separate the protein coats from the bacterial cells. So they centrifuged this mixture to get rid of the protein coats. Let's get rid of them. Notice how the phages inside this bacterial cell, they just have a regular protein coat that's not radioactivelylabeled with s35. That's why they're drawn in green. Now what they did was, they took these bacterial cells, lysed them, which means they made them burst, and they analyzed the viruses. They saw that the viruses were not at all radioactivelylabeled. So they concluded that the protein coat must have remained outside the cell, outside the bacterial cell. If the protein coat remained outside of the bacterial cell, then it must be that that is not genetic material, because, in order for this bacterial cell to have produced viruses, it had to have contained the genetic material of the virus. If the protein coat remained outside, it must be that that is not the genetic material. Let's talk about the second part of Hershey and Chase's experiment. Again, they took a phage, or a couple of phages, and put it in a medium with a lot of nutrients so that it can reproduce and make lots of viruses. But this time, they wanted to label not the protein coat but the nucleic acid inside. Again, I'm just gonna refer to nucleic acid as DNA, but keep in mind that it can also be RNA. They wanna label the DNA, and how are they gonna do that? They made sure that all of the nucleotides that were in the broth ... and the viruses are gonna need nucleotides to make their DNA. All of them were radioactively labeled with phosphorus32. P32 is radioactive isotope of phosphorus, and this is how they're gonna label the DNA. The reason they chose phosphorus32 is because phosphorus is found in DNA, in nucleotides, but it is not found in amino acids. So it will not get integrated into the protein coat. It's a great way to differentiate between the two. They put them in the broth in a lab and to reproduce, and they produce a generation of viruses that have this radioactivelylabeled DNA, which I drew in that magenta. Of course, the protein coat is in green because it's not labeled in any way. They allowed these viruses, these phages, to infect the bacterial cell. The protein coat remains outside and it injects the DNA into the bacterial cell, and the bacterial cell is going to produce a whole bunch of phages. The protein coats, of course, remain outside. Now they're gonna centrifuge this mixture of protein coats and bacterial cells because they want to get rid of the protein coats. So they spin it in a centrifuge and the protein coats, which are less than (mumbles), will end up in a supernatant and the bacterial cells, which are heavier, end up in the pellet. We're gonna get rid ... Sorry about that. We're gonna get rid of these protein coats. Now Hershey and Chase lysed these bacterial cells to make them burst and they analyze the viruses inside. The viruses have a lot of nucleotides with this p32, so there's a lot of radioactivelylabeled DNA. Maybe not all the DNA had radioactivelylabeled phosphorus, because some of the nutrients came from the bacterial cell, but a fair number of the viruses had radioactivelylabeled DNA inside of them. They concluded that, since the DNA entered the cell, it must be DNA that's genetic material. Or, it should really be more specific. It's nucleic acid that's genetic material. In order for the bacterial cell to have produced viruses, it had to have inside of it genetic material. Since there's this radioactivelylabeled DNA within the viruses, they concluded that nucleic acid is genetic material. We mentioned that Hershey and Chase published the findings of their experiment in 1952. A very, very short while later, in 1953, James Watson and Francis Crick published their famous paper in which they actually identified the structure of DNA, or nucleic acids. They put together a tremendous amount of research that was happening during their time and before their time and they identified the structure of DNA. They told us that DNA is a doublestranded helix with a sugarphosphate backbone, the sugar in this case being deoxyribose. Then what you see on the inside are kind of what looks like the rungs of a ladder. Those are nitrogen bases. There are four of them, adenine, thymine, guanine and cytosine. Adenine and thymine pair up with each other and guanine and cytosine pair up with each other. Let's just recap the four experiments that we discussed. We spoke about Friedrich Miescher. Miescher was the first one to isolate and identify nucleic acids. Then we spoke about Wilhelm Roux. Roux's experiments helped show that it was the material in the nucleus that was genetic material. But still, at that point, people weren't quite sure if it's protein or DNA. Many people thought it was protein, because proteins are more complex than nucleic acids. Then we spoke about Hershey and Chase and how they helped prove that is was nucleic acid that's genetic material and not protein. Then Watson and Crick actually identified the structure of nucleic acids. |
78 | Biomolecules | Chromosomal Inheritance | Sex-linked traits | By this point in the biology playlist, you're probably wondering a very natural question, how is gender determined in an organism? And it's not an obvious answer, because throughout the animal kingdom, it's actually determined in different ways. In some creatures, especially some types of reptiles, it's environmental. Not all reptiles, but certain cases of it. It could be maybe the temperature in which the embryo develops will dictate whether it turns into a male or female or other environmental factors. And in other types of animals, especially mammals, of which we are one example, it's a genetic basis. And so your next question is, hey, Sal, so let me write this down, in mammals it's genetic so, OK, maybe they're different alleles, a male or a female allele. But then you're like, hey, but there's so many different characteristics that differentiate a man from a woman. Maybe it would have to be a whole set of genes that have to work together. And to some degree, your second answer would be more correct. It's even more than just a set of genes. It's actually whole chromosomes determine it. So let me draw a nucleus. That's going to be my nucleus. And this is going to be the nucleus for a man. So 22 of the pairs of chromosomes are just regular nonsexdetermining chromosomes. So I could just do, that's one of the homologous, 2, 4, 6, 8, 10, 12, 14. I can just keep going. And eventually you have 22 pairs. So these 22 pairs right there, they're called autosomal. And those are just our standard pairs of chromosomes that code for different things. Each of these right here is a homologous pair, homologous, which we learned before you get one from each of your parents. They don't necessarily code for the same thing, for the same versions of the genes, but they code for the same genes. If eye color is on this gene, it's also on that gene, on the other gene of the homologous pair. Although you might have different versions of eye color on either one and that determines what you display. But these are just kind of the standard genes that have nothing to do with our gender. And then you have these two other special chromosomes. I'll do this one. It'll be a long brown one, and then I'll do a short blue one. And the first thing you'll notice is that they don't look homologous. How could they code for the same thing when the blue one is short and the brown one's long? And that's true. They aren't homologous. And these we'll call our sexdetermining chromosomes. And the long one right here, it's been the convention to call that the x chromosome. Let me scroll down a little bit. And the blue one right there, we refer to that as the y chromosome. And to figure out whether something is a male or a female, it's a pretty simple system. If you've got a y chromosome, you are a male. So let me write that down. So this nucleus that I drew just here obviously you could have the whole broader cell all around here this is the nucleus for a man. So if you have an x chromosome and we'll talk about in a second why you can only get that from your mom an x chromosome from your mom and a y chromosome from your dad, you will be a male. If you get an x chromosome from your mom and an x chromosome from your dad, you're going to be a female. And so we could actually even draw a Punnett square. This is almost a trivially easy Punnett square, but it kind of shows what all of the different possibilities are. So let's say this is your mom's genotype for her sexdetermining chromosome. She's got two x's. That's what makes her your mom and not your dad. And then your dad has an x and a y I should do it in capital and has a Y chromosome. And we can do a Punnett square. What are all the different combinations of offspring? Well, your mom could give this X chromosome, in conjunction with this X chromosome from your dad. This would produce a female. Your mom could give this other X chromosome with that X chromosome. That would be a female as well. Well, your mom's always going to be donating an X chromosome. And then your dad is going to donate either the X or the Y. So in this case, it'll be the Y chromosome. So these would be female, and those would be male. And it works out nicely that half are female and half are male. But a very interesting and somewhat ironic fact might pop out at you when you see this. Who determines whether their offspring are male or female? Is it the mom or the dad? Well, the mom always donates an X chromosome, so in no way does what the haploid genetic makeup of the mom's eggs, of the gamete from the female, in no way does that determine the gender of the offspring. It's all determined by whether let me just draw a bunch of dad's got a lot of sperm, and they're all racing towards the egg. And some of them have an X chromosome in them and some of them have a Y chromosome in them. And obviously they have others. And obviously if this guy up here wins the race. Or maybe I should say this girl. If she wins the race, then the fertilized egg will develop into a female. If this sperm wins the race, then the fertilized egg will develop into a male. And the reason why I said it's ironic is throughout history, and probably the most famous example of this is Henry the VIII. I mean it's not just the case with kings. It's probably true, because most of our civilization is male dominated, that you've had these men who are obsessed with producing a male heir to kind of take over the family name. And, in the case of Henry the VIII, take over a country. And they become very disappointed and they tend to blame their wives when the wives keep producing females, but it's all their fault. Henry the VIII, I mean the most famous case was with Ann Boleyn. I'm not an expert here, but the general notion is that he became upset with her that she wasn't producing a male heir. And then he found a reason to get her essentially decapitated, even though it was all his fault. He was maybe producing a lot more sperm that looked like that than was looking like this. He eventually does produce a male heir so he was and if we assume that it was his child then obviously he was producing some of these, but for the most part, it was all Henry the VIII's fault. So that's why I say there's a little bit of irony here. Is that the people doing the blame are the people to blame for the lack of a male heir. Now one question that might immediately pop up in your head is, Sal, is everything on these chromosomes related to just our sexdetermining traits or are there other stuff on them? So let me draw some chromosomes. So let's say that's an X chromosome and this is a Y chromosome. Now the X chromosome, it does code for a lot more things, although it is kind of famously gene poor. It codes for on the order of 1,500 genes. And the Y chromosome, it's the most gene poor of all the chromosomes. It only codes for on the order of 78 genes. I just looked this up, but who knows if it's exactly 78. But what it tells you is it does very little other than determining what the gender is. And the way it determines that, it does have one gene on it called the SRY gene. You don't have to know that. SRY, that plays a role in the development of testes or the male sexual organ. So if you have this around, this gene right here can start coding for things that will eventually lead to the development of the testicles. And if you don't have that around, that won't happen, so you'll end up with a female. And I'm making gross oversimplifications here. But everything I've dealt with so far, OK, this clearly plays a role in determining sex. But you do have other traits on these genes. And the famous cases all deal with specific disorders. So, for example, color blindness. The genes, or the mutations I should say. So the mutations that cause color blindness. Redgreen color blindness, which I did in green, which is maybe a little bit inappropriate. Color blindness and also hemophilia. This is an inability of your blood to clot. Actually, there's several types of hemophilia. But hemophilia is an inability for your blood to clot properly. And both of these are mutations on the X chromosome. And they're recessive mutations. So what does that mean? It means both of your X chromosomes have to have let's take the case for hemophilia both of your X chromosomes have to have the hemophilia mutation in order for you to show the phenotype of having hemophilia. So, for example, if there's a woman, and let's say this is her genotype. She has one regular X chromosome and then she has one X chromosome that has the I'll put a little superscript there for hemophilia she has the hemophilia mutation. She's just going to be a carrier. Her phenotype right here is going to be no hemophilia. She'll have no problem clotting her blood. The only way that a woman could be a hemophiliac is if she gets two versions of this, because this is a recessive mutation. Now this individual will have hemophilia. Now men, they only have one X chromosome. So for a man to exhibit hemophilia, to have this phenotype, he just needs it only on the one X chromosome he has. And then the other one's a Y chromosome. So this man will have hemophilia. So a natural question should be arising is, hey, you know this guy let's just say that this is a relatively infrequent mutation that arises on an X chromosome the question is who's more likely to have hemophilia? A male or a female? All else equal, who's more likely to have it? Well if this is a relatively infrequent allele, a female, in order to display it, has to get two versions of it. So let's say that the frequency of it and I looked it up before this video roughly they say between 1 in 5,000 to 10,000 men exhibit hemophilia. So let's say that the allele frequency of this is 1 in 7,000, the frequency of Xh, the hemophilia version of the X chromosome. And that's why 1 in 7,000 men display it, because it's completely determined whether there's a 1 in 7,000 chance that this X chromosome they get is the hemophilia version. Who cares what the Y chromosome they get is, cause that essentially doesn't code at all for the blood clotting factors and all of the things that drive hemophilia. Now, for a woman to get hemophilia, what has to happen? She has to have two X chromosomes with the mutation. Well the probability of each of them having the mutation is 1 in 7,000. So the probability of her having hemophilia is 1 in 7,000 times 1 in 7,000, or that's 1 in what, 49 million. So as you can imagine, the incidence of hemophilia in women is much lower than the incidence of hemophilia in men. And in general for any sexlinked trait, if it's recessive, if it's a recessive sexlinked trait, which means men, if they have it, they're going to show it, because they don't have another X chromosome to dominate it. Or for women to show it, she has to have both versions of it. The incidence in men is going to be, so let's say that m is the incidence in men. I'm spelling badly. Then the incidence in women will be what? You could view this as the allele frequency of that mutation on the X chromosome. So women have to get two versions of it. So the woman's frequency is m squared. And you might say, hey, that looks like a bigger number. I'm squaring it. But you have to remember that these numbers, the frequency is less than 1, so in the case of hemophilia, that was 1 in 7,000. So if you square 1 in 7,000, you get 1 in 49 million. Anyway, hopefully you found that interesting and now you know how we all become men and women. And even better you know whom to blame when some of these, I guess, malefocused parents are having trouble getting their son. |
79 | Biomolecules | Chromosomal Inheritance | Worked example: Punnett squares | In the last video, I drew this grid in order to understand better the different combinations of alleles I could get from my mom or my dad. And this grid that I drew is called a Punnett square. And I looked up what Punnett means, and it turns out, and this might be the biggest takeaway from this video, that when you go to the farmers' market or you go to the produce and you see those little baskets, you see those little baskets that often you'll see maybe strawberries or blueberries sitting in, they have this little grid here, right there. Sometimes grapes are in them, and you have a bunch of strawberries in them like that. That green basket is a punnett. That's a punnett. Apparently, in some countries, they call it a punnett. I think England's one of them, and you UK viewers can correct me if I'm wrong. And so I guess that's where the inspiration comes for calling these Punnett squares, that these are kind of these little green baskets that you can throw different combinations of genotypes in. And these Punnett squares aren't just useful. If you're talking about crossing two hybrids, this is called a monohybrid cross because you are crossing two hybrids for only one trait. It could be useful for a whole set of different types of crosses between two reproducing organisms. It doesn't even have to be a situation where one thing is dominating another. Let's do a bunch of these, just to make you familiar with the idea. So let's say you have a mom. So instead of doing two hybrids, let's say the mom I'll keep using the blueeyed, browneyed analogy just because we're already reasonably useful to it. Let's say that she's homozygous dominant. And let's say that the dad is a heterozygote, so he's got a brown and he's got a blue. And we want to know the different combinations of genotypes that one of their children might have. So what we do is we draw a Punnett square again. Let me draw a grid here and draw a grid right there. And up here, we'll write the different genes that mom can contribute, and here, we'll write the different genes that dad can contribute, or the different alleles. I didn't want to write gene. I wanted to write dad. So the mom in either case is either going to contribute this big B brown allele from one of the homologous chromosomes, or on the other homologous, well, they have the same allele so she's going to contribute that one to her child. The dad could contribute this one, that big browneyed the capital B allele for brown eyes or the lowercase b for blue eyes, either one. So the different combinations that might happen, an offspring could get both of these brown alleles from one copy from both parents. This could also happen where you get this brown allele from the dad and then the other brown allele from the mom, or you could get a brown allele from the mom and a blueeyed allele from the dad, or you could get the other browneyed allele from the mom, right? When the mom has this, she has two chromosomes, homologous chromosomes. Each of them have the same brown allele on them. They both have that same brown allele, so I could get the other one from my mom and still get this blueeyed allele from my dad. So if you said what's the probability of having a blueeyed child, assuming that blue eyes are recessive? And remember, this is a phenotype. These particular combinations are genotypes. Well, in order to have blue eyes, you have to be homozygous recessive. You have to have two lowercase b's. So what's the probability of having this? Well, there are no combinations that result in that, so there's a 0% probability of having two blueeyed children. What's the probability of having a homozygous dominant child? Let me write that. A homozygous dominant. And now we're looking at the genotype. We care about the specific alleles that that child inherits. Well, which of these are homozygous dominant? Well, you have this one right here and you have that one right there, and so two of the four equally likely combinations are homozygous dominant, so you have a 50% shot. And we can do these Punnett squares. They don't even have to be for situations where one trait is necessarily dominant on the other. For example, you could have the situation it's called incomplete dominance. Let's say you have two traits for color in a flower. You could have red flowers or you could have white flowers. And let's say I were to cross a parent flower that has the genotype capital R I'll just make it in a capital W. So that could be the mom or the dad, although the analogy breaks down a little bit with parents, although there is a male and female, although sometimes on the same plant. And let's say the other plant is also a red and white. The other plant has a red allele and also has a white allele. So what are the different possibilities? Well, we just draw our Punnett square again. Let me draw our little grid. So the child could inherit both of these red alleles. He could inherit this white allele and then this red allele, so this red one and then this white one, right? That's that right there and that red one is that right there. Or it could inherit this red one from let's say this is the mom plant and then the white allele from the dad plant, so that's that one right there. Or you could inherit both white alleles. What I said when I went into this, and I wrote it at the top right here, is we're studying a situation dealing with incomplete dominance. So what does that mean? Well, that means you might actually have mixing or blending of the traits when you actually look at them. So if this was complete dominance, if red was dominant to white, then you'd say, OK, all of these guys are going to be red and only this guy right here is going to be white, so you have a one in four probability to being white. But let's say that a heterozygous genotype so let me write that down. Let's say when you have one R allele and one white allele, that this doesn't result in red. This results in pink. So this is what blending is. It's kind of a mixture of the two. So if I said if these these two plants were to reproduce, and the traits for red and white petals, I guess we could say, are incomplete dominant, or incompletely dominant, or they blend, and if I were to say what's the probability of having a pink plant? And now when I'm talking about pink, this, of course, is a phenotype. So the probability of pink, well, let's look at the different combinations. How many of these are pink? This one is pink and this is pink. So two are pink of a total of four equally likely combinations, so it's a 50% chance that we're pink. And we could keep doing this over multiple generations, and say, oh, what happens in the second and third and the fourth generation? Actually, we could even have a situation where we have multiple different alleles, and I'll use almost a kind of a more realistic example. I'll use blood types as an example. So there's three potential alleles for blood type. You can have a blood type A, you could have a blood type B, or you could have a blood type O. What happens is you have a combination here between codominance and recessive genes. And I'm going to show you what I talk about when we do the Punnett squares. Maybe I'll stick to one color here because I think you're getting the idea. So let's say I have a parent who is AB. So that means that they have on one of their homologous chromosomes, they have the A allele, and on the other one, they have the B allele. That's what AB means. So the phenotype is the genotype. They're codominant. They both express themselves. They don't necessarily blend. They both express. That's an AB blood type. Let me write this right here. This is AB blood type. And then the other parent is let's say that they are fully an A blood type. Let's say they're an A blood type. Let's say their phenotype is an A blood type I hope I'm not confusing you but their genotype is that they have one allele that's an A and their other allele that's an O. So this is what's interesting about blood types. It's a mixture. O is recessive. O is recessive, while these guys are codominant. So if you have either of these guys with an O, these guys dominate. If you have them together, then your blood type is AB. So what are all the different combinations for these for this couple here? Well, you could get this A and that A, so you get an A from your mom and you get an A from your dad right there. And clearly in this case, your phenotype, you will have an A blood type in this situation. You could get the A from your dad and you could get the B from your mom, in which case you have an AB blood type. You could get the A from your mom and the O from your dad, in which case you have an A blood type because this dominates that. Or you could get the B from your I dont want to introduce arbitrary colors. You could get the B from your mom, that's this one, or the O from your dad. No, once again, I introduced a different color. And this is a B blood type. So if I said what's the probability of having an AA blood type? And once again, we're talking about a phenotype here. So which of these are an A blood type? This one definitely is, because it's AA. If you have two A alleles, you'll definitely have an A blood type, but you also have an A blood type phenotype if you have an A and then an O. O is recessive. So this is also going to be an A blood type. So these are both A blood, so there's a 50% chance, because two of the four combinations show us an A blood type. And you could do all of the different combinations. You say, well, how do you have an O blood type? Well, both of your parents will have to carry at least one O. So, for example, to have a that would've been possible if maybe instead of an AB, this right here was an O, then this combination would've been two O's right there. So hopefully, that gives you an idea of how a Punnett square can be useful, and it can even be useful when we're talking about more than one trait. So let's go to our situation that I talked about before where I said you have little b is equal to blue eyes, and we're assuming that that's recessive, and you have big B is equal to brown eyes, and we're assuming that this is dominant. And let's say we have another trait. I introduced that tooth trait before. So let's say little t is equal to small teeth. I don't know what type of bizarre organism I'm talking about, although I think I would fall into the big tooth camp. Let's say big T is equal to big teeth. So an individual can have for example, I might be heterozygous brown eyes, so my genotype might be heterozygous for brown eyes and then homozygous dominant for teeth. So this might be my genotype. And the phenotype for this one would be a bigtoothed, browneyed person, right? Let me make that clear. This is big tooth phenotype. And this is the phenotype. What you see is brown eyes. A bigtoothed, browneyed person. Now if we assume that the genes that code for teeth or eye color are on different chromosomes, and this is a key assumption, we can say that they assort independently. Let me write that down: independent assortment. So this is a case where if I were look at my chromosomes, let's say this is one homologous pair, maybe we call that homologous pair 1, and let's say I have another homologous pair, and obviously we have 23 of these, but let's say this is homologous pair 2 right here, if the eye color gene is here and here, remember both homologous chromosomes code for the same genes. They might have different versions. Those are alleles. And if teeth are over here, they will assort independently. So after meiosis occurs to produce the gametes, the offspring might get this chromosome or a copy of that chromosome for eye color and might get a copy of this chromosome for teeth size or tooth size. Or it could go the other way. Maybe another offspring gets this one, this chromosome for eye color, and then this chromosome for teeth color and gets the other version of the allele. So because they're on different chromosomes, there's no linkage between if you inherit this one, whether you inherit big teeth, whether you're going to inherit small brown eyes or blue eyes. Now, if they were on the same chromosomee let's say the situation where they are on the same chromosome. So let me pick another trait: hair color. Let's say the gene for hair color is on chromosome 1, so let's say hair color, the gene is there and there. These might be different versions of hair color, different alleles, but the genes are on that same chromosome. In this situation, if someone gets let's say if this is blue eyes here and this is blond hair, then these are going always travel together. You're not going to have these assort independently. And these are called linked traits. Let me highlight that. So these right there, those are linked traits. But for a second, and we'll talk more about linked traits, and especially sexlinked traits in probably the next video or a few videos from now, but let's assume that we're talking about traits that assort independently, and we cross two hybrids. So this is called a dihybrid cross. Very fancy word, but it just gives you an idea of the power of the Punnett square. So let's say both parents are so they're both hybrids, which means that they both have the dominant browneye allele and they have the recessive blueeye allele, and they both have the dominant bigtooth gene and they both have the recessive little tooth gene. So this is the genotype for both parents. Both parents are dihybrid. They're hybrids for both genes, both parents. What are all the different combinations for their children? And I could have done this without dihybrids. I could have made one of them homozygous for one of the traits and a hybrid for the other, and I could have done every different combination, but I'll do the dihybrid, because it leads to a lot of our variety, and you'll often see this in classes. So if I'm talking about the mom, what are the different combinations of genes that the mom can contribute? Well, the mom could contribute the brown so for each of these traits, she can only contribute one of the alleles. So she could contribute this brown right here and then the big yellow T, so this is one combination, or she could contribute the big brown and then the little yellow t, or she can contribute the blueeyed allele and the big T. So these are all the different combinations that she could contribute. And then the final combination is this allele and that allele, so the blue eyes and the small teeth. So that's from mom. And, of course, dad could contribute the same different combinations because dad has the same genotype. Let me write that down. Let me just write it like this so I don't have to keep switching colors. Actually, I want to make them a little closer together because I'm going to run out of space otherwise. Nope. Let me do it like that. OK, brown eyes, so the dad could contribute the big teeth or the little teeth, z along with the browneyed gene, or he could contribute the blueeyed gene, the blueeyed allele in combination with the big teeth or the yellow teeth. Not the yellow teeth, the little teeth. That would be a different gene for yellow teeth or maybe that's an environmental factor. So these are all the different combinations that can occur for their offspring. So let's draw call this maybe a super Punnett square, because we're now dealing with, instead of four combinations, we have 16 combinations. It looks like I ran out of ink right there. It's strange why 16 combinations. Let me write that out. Something's wrong with my tablet. Maybe there's something weird. OK, so there's 16 different combinations, and let's write them all out, and I'll just stay in one maybe neutral color so I don't have to keep switching. I could get this combination, so this brown eyes from my mom, brown eyes from my dad allele, so its brownbrown, and then big teeth from both. I could have this combination, so I have capital B and a capital B. And then I have a capital T and a lowercase t. And then let's just keep moving forward. So I could get a capital B and a lowercase B with a capital T and a capital T, a big B, lowercase B, capital T lowercase t. And I'm just going to go through these superfast because it's going to take forever, so capital B from here, capital B from there; capital T, lowercase t from here; capital B from each and then lowercase t from each. You have a capital B and then a lowercase b from that one, and then a capital T from the mom, lowercase t from the dad. Hopefully, you're not getting too tired here. And so then you have the capital B from your dad and then lowercase b from your mom. Two lowercase t's actually let me just pause and fill these in because I don't want to waste your time. There I have saved you some time and I've filled in every combination similar to what happens on many cooking shows. But now that I've filled in all the different combinations, we can talk a little bit about the different phenotypes that might be expressed from this dihybrid cross. For example, how many of these are going to exhibit brown eyes and big teeth? So big teeth, browneyed kids. Let me write this down here. So if I want big teeth and brown eyes. All of a sudden, my pen doesn't brown eyes. So how many are there? Big teeth and brown eyes. So they're both dominant, so if you have either a capital B or a capital T in any of them, you're going to have big teeth and brown eyes, so this is big teeth and brown eyes. Big teeth right here, brown eyes there. Or maybe I should just say brown eyes and big teeth because that's the order that I wrote it right here. Brown eyes and big teeth, brown eyes and big teeth. Even though I have a recessive trait here, the brown eyes dominate. I had a small teeth here, but the big teeth dominate. This is brown eyes and big teeth. This is brown eyes and big teeth. Let's see, this is brown eyes and big teeth, brown eyes and big teeth, and let me see, is that all of them? Well, no. This is brown eyes and little teeth. This is brown eyes and big teeth right there, and this is also brown eyes and big teeth. They're heterozygous for each trait, but both brown eyes and big teeth are dominant, so these are all phenotypes of brown eyes and big teeth. So how many of those do we have? We have one, two, three, four, five, six, seven, eight, nine of those. So we have nine. Nine brown eyes and big teeth. Now, how many do we have of big teeth? Let me write in a different color, so let me write brown eyes and little teeth. Something on my pen tablet doesn't work quite right over there. So brown eyes and little teeth. So let's see, this is brown eyes and little teeth right there. This is brown eyes and little teeth right there. This is brown eyes and little teeth right there. So there's three combinations of brown eyes and little teeth. And if I were to say blue eyes, blue and big teeth, what are the combinations there? Well, this is blue eyes and big teeth, blue eyes and big teeth, blue eyes and big teeth, so there's three combinations there. And if I want to be recessive on both traits, so if I want let me do this. I want blue eyes, blue and little teeth. There's only one. Out of the 16, there's only one situation where I inherit the recessive trait from both parents for both traits. So if you look at this, and you say, hey, what's the probability there's only one of that what's the probability of having a big teeth, browneyed child? And these are all the phenotypes. There were 16 different possibilities here, right? There are 16 squares here, and 9 of them describe the phenotype of big teeth and brown eyes, so there's a 9/16 chance. So it's 9 out of 16 chance of having a big teeth, browneyed child. What's the probability of a blueeyed child with little teeth? 1 in 16. So hopefully, in this video, you've appreciated the power of the Punnett square, that it's a useful way to explore every different combination of all the genes, and it doesn't have to be only one trait. It can be in this case where you're doing two traits that show dominance, but they assort independently because they're on different chromosomes. You could use it where'd I do it over here? You could use it to explore incomplete dominance when there's blending, where red and white made pink genes, or you can even use it when there's codominance and when you have multiple alleles, where it's not just two different versions of the genes, there's actually three different versions. So hopefully, you've enjoyed that. |
80 | Biomolecules | Chromosomal Inheritance | Genetic recombination | Let's take a look at the nucleus of a cell that's just starting to undergo meiosis. So we have these 46 chromosomes and beforehand each chromosome probably looked something like that. That's the centromere in middle. And then, as meiosis is beginning, they will duplicate into, each chromosome will duplicate into something like that. That's again that centromere in middle. So we have these 23 purple chromosomes, we're gonna say that these are the maternal chromosomes. And then we're gonna say that the blue ones are the paternal chromosomes. They come in homologous pairs. And the nucleus that we're looking at must belong to the cell of a male. because right over here you can see that is the Y chromosome. It's a bit smaller than most of the other chromosomes. I'm gonna digress just for a moment, to clarify a very common point of confusion. And that is that if you look over here at these two chromosomes, well, this is considered one chromosome, but so is this, called one chromosome. And this can be confusing because this is only one chromatid and this is two chromatids. So why are we calling them each one chromosome? And the answer to that question is because we count chromosomes by the number of centromeres. So this single chromatid has one centromere, but these two chromatids are attached in the middle by one centromere. So we also call that one chromosome. Just keep that in mind, it's just a technical point to know that even though they're different, they're still both considered one chromosome. And in this chromosome, the two chromatids are duplicates of each other. So it's just a copy of itself. Anyway, back to our nucleus. So we have these 46 chromosomes, 23 homologous pairs. And they're not, the chromosomes are not necessarily arranged in the way that I drew them. I just drew it that way for the sake of organization. But, we have these pairs and we're gonna focus on one pair of homologous chromosomes. But I want you to keep in mind throughout this video, that whatever we're describing that's happening to this pair of homologous chromosomes, is also happening to the other 22 pairs of homologous chromosomes. It's just that it would be too hard to depict in a video how that's happening, but keep in mind it's not just happening to the pair that we're talking about, but what we're going to be describing is happening to all of the pairs of chromosomes in the nucleus. So here we have our pair of homologous chromosomes. And during prophase one of meiosis, the homologous chromosomes pair up with each other and form a unit called a tetrad. And it's called a tetrad because, well, tetra means four and this unit has four chromatids, right. One, two, three, and four. And the process during which the homologous chromosomes pair up with each other is called synapsis. So during synapsis, the homologous chromosomes will get a little bit closer to each other. Something like that. And at a certain spot, they might actually cross over or overlap. So I'm gonna circle that spot. And that's called the chiasma. And in some cases, another thing happens. This protein complex that resembles something like a railroad track forms. We'll see in a minute why. And this is called the synaptonemal complex. You can actually see the word synapse in there because this happens during synapsis. So we've formed the synaptonemal complex and with the help of the synaptonemal complex, these two chromatids, the ones that are crossing over, will actually swap material downward of that point. So we're gonna get something that looks like that. Look at how the purple chromosome now has some blue over there. And look at how the blue chromosome now has some purple over there. And the way that happened was that the DNA in the chromosome, actually some bonds in that DNA broke and the DNAs just kinda swapped places. So what we just described, this process by which the two chromosomes swap information is called crossing over. Or, another way to say this, is genetic recombination. And let's see why this is called genetic recombination. So we're gonna fast forward to the end of meiosis to where the chromosomes get split into two and all the chromatids get separated into different gametes. And I want to pause and remind you that everything we're describing that's happening to this pair of chromosomes is also happening to all the other 22 pairs of homologous chromosomes. But anyway, so now let's put each one of the chromatids in a different gamete. And look at how we get four different gametes. And we can call these two, gametes recombinant. And we're calling them recombinant because they have a combination of alleles that's new. We haven't had this combination of alleles, even in a parent. And just to clarify things, let's see what the gametes would look like if crossing over did not happen. So let's go back to our original chromosomes. And let's split them. And let's put them into four different gametes. And we are going to get that. And you can see that in this case, we only have two different types of gametes. So we can see how genetic recombination increases genetic variability. Which is usually a good thing. |
81 | Biomolecules | Chromosomal Inheritance | Gene mapping | One of the very interesting things about genetic recombination is that you can actually use genetic recombination to figure out the distance between genes on a chromosome. And if you were to do this to all the genes on a chromosome, you could actually map out the chromosome, figure out exactly where the genes are. And we're going to explore that concept. So we're looking at a pair of homologous chromosomes, and let's just say that the orange chromosome is the paternal chromosome, so it comes from the father, and let's say that the yellow one is the maternal chromosome. And, just to remind ourselves, these are sister chromatids. That means that they are identical chromatids, they have identical genes on them. So those colored circles that I drew represent just some genes that I randomly picked. And you can see that on the two sister chromatids I drew them in the same color, because they are the same. And for that matter, these are also sister chromatids. These two yellow chromatids are also sister chromatids. And I'm just gonna review a bit to give us some context. So normally in the cell, the paternal chromosome would just look something like that, with the centromere middle. And the maternal chromosome would also look something like that. But, during myosis or mitosis, when they replicate, this chromosome will turn into that. So it's gonna duplicate itself and each one of those strands is a sister chromatid. And so the maternal chromosome turns into something that looks like that. But, this is still considered one chromosome. And this is considered one chromosome. And that can be a little bit confusing, because how could this be one chromosome and this also be one chromosome? If this X has double the material as the original strand? And the answer to that question is because we count chromosomes by the amount of centromeres that are present. So this has one centromere, it's considered one chromosome. This X has one centromere, so it's considered one chromosome. However, they're not quite the same. This, on top, is just one chromatid. And the bottom chromosome has ended up with two chromatids. So try not to get confused, even though we're calling this one chromosome, it's really made up of two identical sister chromatids. But anyway, that was a bit of a digression. Let's focus again on our homologous chromosomes. So I picked a couple of genes, that I just put on, and we're gonna focus on three genes in particular. And to make this a bit more real or relevant, let's just say that the green genes represent I don't know, maybe complexion. Maybe like a dark complexion versus a lighter complexion. And on the maternal chromosome, I also drew it in green, to remind us that these are homologous chromosomes, they're homologous alleles, in other words, these are both alleles that code for complexion, but different shades of green, because they're probably different versions. So these green genes also code for complexion. And let's say that this darker purple, or magenta, gene codes for, let's say hair color. So that means that the lighter purple on the maternal chromosome also code for hair color. And then let's say that the blue genes code for eye color. So we have dark blue on the paternal chromosome and light blue on the maternal chromosome. And let's then focus on the sister chromatids where genetic recombination occurs. So these two strands are going to swap genetic information between them. Maybe like a chunk on bottom will swap, maybe something in middle will swap, maybe something on top will swap. Actually, genetic recombination also occurs between sister chromatids. However, sister chromatids are identical so it would be of no consequence. Anyway, so let's look at the two chromatids where genetic recombination is happening. The two that I circled. Let's take a closer look at those. So here are the two chromatids that are going to exchange genetic information and undergo recombination. And I want to ask you a question. So I'm gonna give you two choices. And I want you to try to figure out which one is more likely. So, for the first choice, let's look at the purple and green genes. And the question is, what's the what's more likely? Is it more likely that the purple and green genes undergo recombination? And when I say that, I mean that the genes that are originally on one chromosome separate. So I mean to say that, for example, this purple gene will get separated from that gene, and this lighter purple gene will get separated from that gene. So is it more likely that the purple and green genes recombine? Or the way I like to view it, get separated. Or, is it more likely that the blue and purple genes recombine or get separated? And the way to think about this is, well look at the distance between them. So the distance between the purple and green genes is something like that. So in order for recombination to occur with respect to the purple and green genes, it would have to happen somewhere over here. Or on the other side, it doesn't matter, it doesn't make a difference. So you have this whole distance, whereas if you wanted recombination to occur with respect to the purple and blue genes, you have a very smaller area to work with. It would have to happen somewhere here. So somewhere along this line of the chromosome. So, that means that it's much more likely for recombination to happen with respect to the purple and green genes. That is more likely because you have this entire distance to work with. If these two chromatids swap genetic information anywhere along this stretch of the chromosome, so the green and purple genes will separate and recombine. And the blue and purple genes are less likely to recombine because recombination would have to occur only in this little sliver of chromosome and that's just smaller than the other part of the chromosome that we were looking at. So, we just learned a very important concept and that is that the further apart two genes are, the more likely it is that they will recombine. I'm not actually not sure if that's the proper way to use the word, but I just use it that way. So I'm gonna put it in quotes. And again, when I say recombine, I mean that two chromosomes that were originally on the same, sorry, two genes that were originally on the same chromosome get separated. And then the next thing we learned is that the closer two genes are to each other, I'll abbreviate each other, just e.o., the less likely it is that they will recombine. And now i'm gonna introduce you to some terms. The centimorgan is the unit of measurement that we use to measure distance on a chromosome. And another way to say centimorgan is a genetic map unit. Or, m.u. which stands for map unit. And I'll give you the official definition of a centimorgan, because I think it's important and it kind of ties in distance to what it has to do with recombination. And so a centimorgan is the distance between genes, I'm just gonna abbreviate between like that, for which one product of myosis in 100 is recombinant. And to put that in simpler terms, it means that if two genes are one centimorgan apart, it means that one out of one hundred times, or one percent of the time that myosis happens, those two genes will be recombinant, or separate, or recombine. And we'll actually do an example of this to illustrate what this means. So here we have our two chromatids again and let's just say that the distance between the purple and green genes is 25 map units. Remember, a map unit is the same thing as a centimorgan. And let's say that the distance between the blue and purple genes is six map units. So this is clearly not drawn to scale very well, but it's just an estimate. So let's first focus on the purple and green genes. So if they're 25 map units apart, so remember if two genes are one map unit apart that means that one percent of the time they'll recombine, so these are 25 map units apart, so that means that 25% of the time that myosis happens, recombination will occur with respect to the purple and green genes. So let's see what that looks like. So we're gonna see that in this spot, right over here, the chromatids just swap information, So let's draw what that would look like. So let's draw our paternal chromosome, or at least part of it. And then we'll draw our, actually I'm gonna draw that a little bit higher, because we need more room. So here's our paternal chromosome. And then our maternal chromosome. Or chromatid. And then, since they kind of swapped in this spot, so I'm gonna draw yellow, right over here. That's the part that came from the maternal chromosome. And then I'm gonna draw orange over here. That's the part that came from the paternal chromosome. And now let's fill in our genes. So the blue genes will just stay where they were. So, we just leave them put. And the same goes for the purple genes. But then, we have that piece of maternal chromosome. So we get that hunter green gene over here. And then we get the lime green gene right over here. So this is what I mean by recombination being a separation of genes. So this purple gene and that lime green gene were on the same chromosome before, but they got separated. They're not on the same chromosome any more. And the same applies to these two genes. Now let's look at the blue and purple genes. So they're six map units apart. So that means that 6% of the time that myosis happens, the purple and blue genes will get separated. So, we're gonna see that upwards of this spot, the chromatids swap information. So, let's draw that. So here we'll draw a part of our paternal. that's the paternal chromosome and the maternal one. And they swapped somewhere like over here. So let's fill that in. So that's the part of the maternal chromosome that lands up, sorry, yeah, that's the part of the maternal chromosome that ends up on the paternal one. And the orange over here. And now let's fill in our genes. So lets's first fill in the ones that just stay put. So we have that lime green gene, then we have that hunter green gene. And our purples also stay put. But the blue genes swap chromosomes. So we have our dark blue gene over here and our light blue gene over here. And again, take note of how they swap places, or how the separate. So these two genes were together on the same chromosome before, but not anymore. And the same for these two genes. So let's just tie this back into the bigger concept going on. If we were to do a statistical analysis of how often certain recombinations happen, that can help us map out the genes on a chromosome. |
82 | Biomolecules | Chromosomal Inheritance | Extranuclear inheritance 1 | Normally when we think about DNA, we think about the nucleus of a cell and that's because a cell's DNA is contained in its nucleus, but there are actually a few exceptions to this general rule. There are certain organelles that actually have their own DNA and two very famous examples of this are the mitochondria and chloroplasts. Mitochondria and chloroplasts have their own DNA, which I'm just going to scribble here in blue, and not only do they have their own DNA but they can actually replicate their DNA and replicate themselves independently of the nucleus of the cell in which they are. Let's just talk briefly about mitochondria. Mitochondria are these organelles found in eucariotic cells and they're sometimes referred to as the "powerhouse" of the cell because they break down glucose to make this highenergy molecule called ATP, and then the cell takes this ATP and uses it for all sorts of cellular processes. And the mitochondrial DNA, written like that "mtDNA", has about 37 genes in it. And these genes, most of them have to do with the cellular respiration that's going on in the mitochondria. Let's talk a bit about chloroplasts. Chloroplasts are these organelles that are found in plant cells. They are also found in algae cells. And chloroplasts are the site of photosynthesis. If we wanted to be more specific, you have these stacks called granum, well in singular, it's granum, plural is grana, and those granum are made up of these... That's an m over there. And those granum are made up of these little circles called thylakoids, and photosynthesis happens within these thylakoids. So during photosynthesis, sunlight is harnessed, of course, with a bunch of other steps to make glucose. This is where the concept of making its own food comes from. It's actually making glucose. It's making its own food. And then, that glucose goes to the mitochondria of that cell and gets broken down, make ATP, and then the cell uses that ATP for whatever it needs to do. The DNA in chloroplast, sometimes are in cpDNA, has about 100 genes and these genes, also, most of them have to do with proteins or things that are involved in photosynthesis. And the reason that this is interesting is, well, let's take a look at how sexual reproduction normally takes place. We have an egg cell and the nucleus of this egg cell has only half the amount of DNA that a normal cell in that organism would have. We call that "n" and then we have a sperm cell. Remember, the sperm cell is really much, much smaller than an egg cell, so this is in no way drawn to scale. And the sperm cell also has in its nucleus, only half the amount of DNA that cells in this organism normally have. That's also "n". But, then they fuse to make a zygote. And this zygote is 2n. It has the normal amount of DNA that a cell in this organism would have. Half of it comes from the egg cell and half of it comes from the sperm cell. And on this zygote is going to divide into two cells and those two cells, of course, divide further and this goes on and on until they are enough cells to put together an organism. But this egg cell, well, it's a fully developed cell and it not only has genetic information, but it has organelles in the cytoplasm. It has these mitochondria in its cytoplasm and those mitochondria have DNA in it, which I'm just going to scribble some blue inside, and these zygote also has those mitochondria, because you remember, the zygote is practically an egg cell with the only difference being that it's nucleus has the additional DNA of the sperm cell. And remember, the sperm cell does not donate anything to the egg cell except for half of the DNA in the nucleus. It does not give the zygote anything else. You have those zygote with those mitochondria, and of course, they have their DNA in it. And then when this zygote replicates itself, so it replicates the nucleus, but it also replicates the mitochondria in the cytoplasm and these cells will... I'm going to skip up the nucleus. I'm just drawing the mitochondria. So I have these mitochondria, but these mitochondria came only from the egg cell and none of those mitochondria came from the sperm cell. And so, this brings us to concept of maternal inheritance. And, maternal inheritance, well it's basically like exactly the way it sounds, it's inheritance that happens only from the maternal line or only from the egg cell. So right here, we're showing that the mitochondria that this organism will eventually have originates from the mitochondria that it came only from the egg cell and not from the sperm cell. And therefore, it exhibits maternal inheritance. So, both mitochondria and chloroplasts exhibit maternal inheritance because they are in the egg cell that eventually becomes the organism. And the maternal inheritance, it's interesting to note, is contrary to mendelian genetics. Maternal inheritance is contrary to mendelian genetics because mendelian genetics assumes that half of the DNA comes from the egg cell, half from the sperm cell, it does not take into account any sort of genetic information that comes from only one of the gametes, for example just from the egg cell, and in fact everything we just described here can be referred to as extranuclear inheritance. Extranuclear inheritance would refer to any genes that are passed on from structures that are not in the nucleus. Extranuclear meaning outside of the nucleus. Mitochondria and chloroplasts are outside of the nucleus. When they are inherited, we refer to it as extranuclear inheritance. Now that we've introduced extranuclear inheritance, let's actually take a look at one of the earlier experiments that helped to discover extranuclear inheritance. |
83 | Biomolecules | Chromosomal Inheritance | Extranuclear inheritance 2 | There was a scientist by the name of Carl Correns. And he was a somewhat of a contemporary of Gregor Mendel. He lived from 1864... to 1933. And Gregor Mendel lived from 1822 to 1884. So they were contemporaries, but Correns was younger than Mendel, and we will soon see that Carl Correns helped discover some things that do not fall to the category of Mendelian genetics. So Carl Correns did a lot of experiments with this plant called the 4 o'clock plant. That's how we know it. Which is what you're looking at, I know it looks a little bit like a tree, but it's supposed to be a plant. And actually the scientific name for the 4 o'clock plant is mirabilis jalapa, not exactly sure how to pronounce that, but anyway. So he did a lot of experiments with the 4 o'clock plant and one very interesting thing about this plant is that you can have within the same plant leaves that are a lot of different colors. So let's say that this is the main branch of our plant. And we're going to say that the leaves that come off anywhere on this branch are going to be white, so there are our white leaves. And we're gonna say that any leaves that come off of this branch are going to be green, so those are our green leaves. And then the leaves that come off of this branch are variegated. And that means that they have a pattern of green and white mixed. So there are our variegated leaves. And why is it that there are all these different colors within the plant? So let's take a look at some of the cells. Let's take a look first at a cell that's from a leaf that's green. So this is in a leaf that's green, so that's our cell. Here's our nucleus. And in case you're wondering why I'm drawing this cell as a square it's because plant cells have this cell wall that give it a more rigid shape and make it a bit more squareish, closer to a square than a circle. But anyway, that's our cell, and this cell will have chloroplasts in it which I'm drawing as these green little circles. And remember chloroplasts have their own DNA, sometimes referred to as cpDNA. And chloroplast DNA has in it basically the stuff that the chloroplasts needs to carry out photosynthesis. And one of the genes in the chloroplast DNA is a gene that makes chlorophyll which is a pigment that's involved in photosynthesis, and chlorophyll is what makes the leaf green. It's a pigment that turns the leaf green or if you wanna be more specific, chlorophyll absorbs all of the colors in sunlight except for green, so green is really reflected, but anyway. The point is that chlorophyll makes the leaf green. So now let's take a look at a cell that comes from a white leaf. So this cell comes from a white leaf, so again we'll draw our nucleus. And then it has chloroplasts, but the chloroplasts in this cell, the DNA in those chloroplasts have a mutation. So the cpDNA has a mutation that does not allow it to produce chlorophyll, or it allows it to produce on a very, very tiny amount of chlorophyll 'cuz it needs chlorophyll to survive, but it's not enough that the leaf will be green. So I'm just gonna write very, very tiny amount of chlorophyll... That's why it appears white. And then the variegated leaves... Well, they have some cells that have regular chloroplasts that make chlorophyll and then they have some cells with the mutated chloroplasts or chloroplasts with mutated DNA that do not make chlorophyll, or rather make a very small amount. And then they actually have a third type of cell... which maybe you're guessing it already, have both types of chloroplasts. They have chloroplasts that do make chlorophyll, and then they have chloroplasts that have mutated DNA and do not make much chlorophyll. And what Correns noticed when he crossed a whole bunch of these plants together, he noticed that the progeny had nothing to do with the sperm cell or the pollen cell. It had only to do with the egg cell. So wherever he would take the seed from, this is where the egg cell's located, if he took it from a branch that only had white leaves, all the progeny had only white leaves. It didn't matter what the pollen cell was. And the same if he took a seed from a branch that had only green leaves, so all the progeny had all green leaves no matter where the pollen cell came from. And this is because the trait that we're looking at, the color of the leaf, well that's determined by the DNA in the chloroplast. And the chloroplast exhibits maternal inheritance. It is going to be inherited only through the egg cell, or through the maternal line, or another way to say this it exhibits extranuclear inheritance... because a chloroplast has DNA that's outside of the nucleus. Let's take a closer look at what Carl Correns did. So we have this chart to help us out. In the first column we have the egg cell of the female, that's the seed. And then we're gonna cross it with what we have in the second column which is the pollen cell, that's the male gamete. And they all look the same because it doesn't make any difference in our case. And then we have in the third column our zygote, or the result. So let's look at our first row. So we have this egg cell that came from a branch that had leaves that were only white. So it came from a flower plant with only white leaves. And when we cross it with a pollen cell, no matter what that pollen cell is, no matter where it came from, we always get the same result. We will always get a plant with only white leaves. Let's look at our second row. So we have this egg cell that came from a branch that had only green leaves. And again, no matter what we cross it with, no matter where the pollen cell came from a white leaf, a variegated leaf, a green leaf, or a flowering green leaf so we always get the same result. We get a plant that has only green leaves. It gets a little bit more interesting when we look at the variegated, at the egg cells from the variegated parts of the plant because there are three different types of egg cells we could have. We could have one that resembles the cell you see if it came from a flower with only white leaves. Then we have another egg cell that looks like that. It looks like the egg cell you'd find in a plant that had only green leaves. And then we have this third type of interesting cell that has the combination of the quote unquote normal chloroplasts that are green, and then it has some chloroplasts that have that mutation that don't allow it to make chlorophyll or make a very small amount of chlorophyll, so it's kind of mixed. Anyway, let's look at egg type one, we cross it with a pollen cell, we always get the same result. You get a plant with only white leaves. And then we look at egg cell type two, whatever we cross it with, we get only green leaves. And then if the egg cell type three, so the zygote of course will have both types of chloroplast, but remember this zygote is going to divide further and it's gonna divide into, if it divides, you know, randomly, it'll divide into three different types of cells. Some of the cells will look like this with the chloroplasts with the mutated DNA, some of them are going to look like that with the regular chloroplasts, and then some of them are going to be mixed. And then this will give you a variegated plant. Some of the leaves are going to have that mixed pattern, you might have some leaves that are white, you might have some leaves that are green. And so the bottom line, take home message, is, as I explained before, because the particular trait we're looking at, leaf color, because the gene for that trait is in the chloroplast, it exhibits maternal inheritance. Maternal inheritance is a type of extranuclear inheritance. I'm just gonna write that in parentheses. Because this inheritance has to with DNA that's outside of the nucleus, but anyway. This exhibits maternal inheritance because it has nothing to do, this particular trait is not passed down through the male, well it's only passed down through the female. And that is, as we explained before, because the chloroplasts are coming only from the egg cell. The sperm cell does not contribute any chloroplasts to the zygote, it only contributes DNA that's in the nucleus. So therefore, the leaf color of this 4 o'clock plant exhibits maternal inheritance. And the same concept would apply to the mitochondria. So we explained mitochondria also has its own DNA, and so if a person were to have a disease that had to do with the DNA inside of the mitochondria, we would know that that person got it from his or her mother and not from his or her father because the mitochondria also exhibits maternal inheritance. There is one more thing about extranuclear inheritance that I want to mention. And that is, why is it that mitochondria and chloroplasts have their own DNA? Is there something that can explain that? And there is... the endosymbiotic theory... seeks to explain why mitochondria and chloroplasts have their own DNA. And this theory tells us that mitochondria and chloroplasts were once independent prokaryotes. So they lived independently, so of course if they're independent, they need to have their own DNA. But eventually, they joined what I'm going to call an ancestral... eukaryotic... cell. And in case you're wondering like, when this happened, we'll say about one and a half billion years ago. So what happened is that the mitochondria and chloroplasts joined an ancenstral eukaryotic cell, and I'm gonna call it "ancestral" because it's not exactly a eukaryotic cell that we'd see today, but it's a cell that would eventually become a eukaryotic cell. We could also call it a host cell because it's gonna host mitochondria and chloroplasts. So let's put them inside. So now they're living in this host cell. And why do you think they would wanna do this? So they're going to live together in symbiosis. And symbiosis is when organisms live together and each one kind of gives the other something and everybody gains something. So an example of this that you might've heard of is in our intestine, in our gut, we have bacteria e. coli, and at first glance it might seem like it's not such a good thing, but it's actually a really good thing because we give the e. coli a warm and cozy place to live, they get some nutrients from us, and in exchange, they make for us vitamin k which is something very useful for us. So that's an example of symbiosis. Each one of the organisms kind of tries to give something and everybody's happy. So what's going on in the host cell? So the host cell gives the mitochondria and chloroplasts a nice place to live and gives them nutrients, and in exchange, the chloroplast makes glucose through photosynthesis, and then the mitochondria takes that glucose and produces ATP. And then that ATP is used as energy, the mitochondria uses some of the ATP, the chloroplasts uses some of the ATP, and of course the host cell uses some of the ATP. So everybody's happy, everybody's getting something. And then eventually this cell, you know, evolved in many different ways, and of course it became the eukaryotic cell that we know of is today, but you know not exactly the way it was because chloroplasts are not found in all eukaryotic cells. It's basically found in plants as in algae, but that's the basic idea. So let's go back to the term endosymbiotic theory. So endo just means "inside" because the mitochondria and chloroplasts began to live inside a host cell. And symbiotic simply refers to symbiosis. I'm just gonna draw an arrow down here, refers to symbiosis because each part, the mitochondria and chloroplast and host cell, everybody's getting something. And so this would explain why mitochondria and chloroplasts have their own DNA because once upon a long time ago, they were independent prokaryotes, and they lived on their own. |
84 | Biomolecules | Evolution Population Dynmaics | Evolution and natural selection | So today we're going to talk about a topic that's very central to the idea of evolution, and that's natural selection. But before we get into that, I want to talk about what evolution isn't. So evolution isn't when some organism like this monkey magically transforms into a human. And it's also not when an organism changes in some way when it's in trouble, like a person growing wings after jumping off a building. And I want to clarify that this is not what we're referring to when we think of evolution. And evolution is a process that occurs to populations of an organism, not individual members. And it occurs over huge amounts of time, and we're talking millions and millions of years for even small changes. So natural selection is one of the forces that ultimately drives evolution, but what is natural selection exactly? Well, why don't we jump right in and look at an example? Let's say it's 10,000 years ago and people survived by hunting and gathering, but they also have to worry about being chased around by wild animals. So in order to survive, these people need to be able to find food. But they also need to be able to escape from predators. Well, let's say that one of the people of these two has a special genetic trait and has slightly longer legs than the other guy. Now, these longer legs put him at an advantage, because his legs are longer and he can run, let's say two times as fast as everyone else. And because of this, he's more likely to survive when a predator like this bear chases him down. So what this also means is that the guy with the long legs is more likely to reach an age where he's old enough to find a mate, reproduce, and have children who would also have this special trait of longer legs because it's genetic. And because he's more likely to have kids than everyone else, over long periods of time soon more and more of the population will have this special trait. Now, let's look at this idea again but a little more deeply. And let's say there are six people in the world and two of them have longer legs than everyone else, And let's say that the ones with the longer legs have a 50% chance of surviving and reproducing while the shorterlegged people have only a 25% chance of surviving and reproducing. So that means one of our two longlegged people and one of our four shortlegged people here will reach an age where they can reproduce, so now these people who survived will each have four children. And naturally, these children will resemble their parents. And the children of longlegged people will also have long legs, and the children of shortlegged people will have short legs. So now in our next generation, we have four people with long legs and four with short legs. And you can already see that more of the population has long legs than when we started. But let's take it another generation further. So half of our longlegged people will reproduce, whereas only 1/4 of our shortlegged people will reproduce. And this means that by our third generation, we'll have eight longlegged kids and only four shortlegged ones. Now, if we number our generations, generations one, two, and three, we can see that in generation one, 33% of the population was long legged. In generation two, 50% of the population was long legged. And by generation three, 67% of our population was long legged. And this is all because that special trait of having longer legs made those people more likely to survive and reproduce than those with short legs, and this is the crux of how natural selection works. So why is it called natural selection in the first place? Well, let's say with our example of the short and longlegged people. Now, we use the word selection because one trait is advantageous over another and is selected to be passed on to future generations more than other traits. On the other hand, selection can also apply to a disadvantageous trait. If we have people who have really short legs and run really slowly, then those people will be selected against and won't pass on traits to offspring as frequently. Now, we use the word natural because there isn't an individual who's physically selecting which traits are good and which ones are bad. It all has to do with whoever has the greatest probability of surviving. There's no one actually doing the selecting except nature itself. Now finally, I just want to point out that natural selection does not apply to acquired characteristics. If a father teaches his son how to hunt and this makes a child more likely to survive, that isn't a trait that's selected for us since it's not genetic and it's not absolutely passed on to children. So that's why we say that natural selection only applies to heritable traits, with heritable traits being any genetic trait. So what did we learn? Well, first we learned about the concept of natural selection and how traits that help an organism survive are more likely to get passed on to offspring. Next we learned that evolution, which is driven by natural selection, occurs to populations, not individuals, and occurs over a huge period of time. And finally, we learned that natural selection only applies to heritable traits, ones that are genetic and passed down from generation to generation. |
85 | Biomolecules | Evolution Population Dynmaics | Fitness and fecundity | So we're going to talk about the concept of fitness and how it relates to natural selection. But first, let's go over what natural selection is. So if you have a member of a population that has a special genetic trait, like being really strong, then that person is more likely to live to an age where he or she can reproduce and pass on that special trait to offspring. Now, this example only really deals with surviving until the age where reproduction is possible and doesn't really deal with the person's ability to actually reproduce once it gets to that age. Now, you should also remember that populations as a whole will evolve by natural selection, and not individual members of a population. So what is fitness? And what does this term mean? Well, fitness refers to an organism's total ability to pass on traits to offspring. And we can look at fitness as a combination of an organism's ability to survive to an age where it can reproduce, as well as its ability to actually reproduce once it gets there. So our strong guy probably has a higher survival rate than the average person, but actually reproduces the same as everyone else. And how well someone can reproduce is determined by that person's fecundity, which is what we're going to talk about today. So what's fecundity, exactly? Well, fecundity refers to how easily and how often an organism can produce offspring. And when you look at asexual reproduction instead of sexual reproduction, fecundity of bacteria like E. coli is determined by how quickly the E. coli cells can divide and increase their population size. When looking at sexual reproduction, fecundity refers to how well an organism can mate with another, and in the case of mammals like humans, carry and ultimately birth offspring. Now, when looking at the fecundity of humans, people often think that it only applies to female fertility since the females are the ones that are actually carrying the children. But fecundity can also be a measure of a male's ability to produce offspring with a female mate. Now, fecundity is directly related to natural selection because, like any other special trait, high fecundity is selected for. So let's look at an example. Let's say we have a population of six people, two of which are red and four of which are blue. Now, the red and the blue people have the same chance of surviving to an age where they can reproduce, which is 50%. And what this means is that one of our red people will survive to an age where they can reproduce and two of our blue people will survive. But the difference between the two is that the red people who survive will each be able to produce four offspring, whereas the blue people who survive will only be able to produce three each. So by our second generation, we have four red people and six blue people. Now, two of our red people, 50%, will survive. And three of the blue people, also 50%, will survive as well. But once again, the surviving red people will each have four offspring, while the surviving blue people will only have three offspring each. So this leaves our third generation with eight red people and nine blue people. If we now number our generations generations 1, 2, and 3, we see that in generation 1, 33% of the population was red, while 67% of the population was blue. In generation 2, 40% were red and 60% were blue. And by generation 3, 47% were red and 53% were blue. And this increases all because the red people had a special trait of higher fecundity, which made them more able to have offspring than the blue people. And this means that fecundity is selected for by natural selection the same way a trait that benefits survival like strength would be. Now, another interesting way that fecundity is selected for has to do with mate selection. Now, when looking for a female mate, many males associate an attractive woman with words like "curvy." And a curvy female would be one that has a healthy and robust body, that was fit for bearing children. And this ability to easily bear children is a direct indicator of high fecundity. So what did we learn? Well, first we learned that natural selection will select for individuals with traits that give them high fitness. And we can divide fitness into traits that will help with survival and traits that will help with reproduction, which are those that increase fecundity. Second, we learned that fecundity is selected for by natural selection, just like any other special trait would be. |
86 | Biomolecules | Evolution Population Dynmaics | Alternative selection | So natural selection isn't the only force that drives evolution. And there are a few alternative forms of selection that also contribute. But before we look at those, let's go over what natural selection is in the first place. And it's the idea that if you have a member of a population that has a special advantageous genetic trait, then that individual is more likely to live to an age where it can reproduce and pass on that special trait to their offspring. Also remember that natural selection selects for individuals with high fitness. And fitness is a measure of an organism's total ability to pass on their genes to their offspring. And it's a combination of an organism's ability to survive to an age where it can reproduce, but also how well that organism can reproduce once it gets to that age. Also remember that populations will evolve by natural selection and not individual members of those populations. So what are natural selection's alternatives? Well, we're going to talk about two today, group selection and also artificial selection. So let's start with group selection. And this is the idea that genetic traits that benefit the population or group as a whole will still be selected for even if they don't directly actually increase the fitness of the individual with the trait. Words like altruism and martyrdom come to mind. And traits that relate to these ideas are what we're talking about when we think of group selection. And these traits can still be selected for because entire populations evolve, not just individual members. So let's look at an example. Let's say a female human has children, and her children have children of their own. Why is it that this female grandmother is able to survive after she becomes unable to have children of her own, let's say when she's already gone through menopause, which is when the female reproductive system shuts down? Any traits that would allow a human to live past this age couldn't be selected for by natural selection since by the time those traits manifested, the person would have already lost their ability to reproduce. Well, it turns out that grandparents play a distinct role in taking care of their grandchildren. And since their care increases the survival rate and thus the fitness of their grandchildren, this helps the group as a whole. And those traits that benefit survival into old age can then still be selected for by group selection. So natural selection will typically look for traits that help a survival until the age where reproduction is possible. But group selection accounts for all those other traits that might help with survival after reproduction is no longer possible. So we talked about the first alternative to natural selection. And this was group selection. But what about artificial selection? Well, in order to find what artificial selection is, let's take a step back and look at natural selection one more time. Remember that if we have a strong individual who is more likely to survive because of his or her strength, then that trait is said to be passed on to offspring more frequently than another trait. And this selection is said to be natural because it all has to do with the idea that the stronger person has a greater probability of surviving than someone else, let's say a 75% chance instead of a 50% chance of surviving. There's no outside individual who's deciding and selecting for which traits are better than others. It all happens naturally. But that's exactly the difference between natural and artificial selection. In fact, some people call artificial selection unnatural selection. And let's explain this by jumping right in with an example. If you have a farmer growing tomatoes, and some tomatoes grow bigger than others, then that farmer can literally select and choose which tomato seeds he uses to plant tomatoes next year. He's artificially selecting tomatoes for those that have a trait which makes them grow more fruit. And that's just one of the many traits of the tomato. Another great example of artificial selection is when scientists in the lab look at a tomato's DNA and again select for specific genes that make the tomato grow larger in order to give the farmer more fruit. And both of these examples, since there's an outside being selecting which traits are desired, we say that the tomatoes are undergoing artificial selection, instead of natural selection, because it doesn't occur naturally. It's not just a matter of probability. So what did we learn? Well, first we learned that natural selection is not the only force driving evolution. We have group selection, which is the idea that traits benefiting the group over the individual with the trait can still be selected for. And we also talked about artificial selection, which is where an outside individual can literally choose which traits in a given population will be passed on, instead of that selection occurring naturally. |
87 | Biomolecules | Evolution Population Dynmaics | Genetic drift, bottleneck effect, and founder effect | We've already made several videos over evolution, and just to remind ourselves what evolution is talking about, it's the change in heritable traits of a population over generations. And a lot of times, you'll hear people say evolution and Natural Selection really in the same breath, but what we wanna make a little bit clear in this video is that Natural Selection is one mechanism of evolution. It's the one most talked about because it is viewed as the primary mechanism. Natural Selection. But what we're gonna talk about in this video is another mechanism called Genetic Drift. So there's Natural Selection, and there is Genetic Drift. Now we've done many videos on Natural Selection, but it's this idea that you have variation in a population, you have different heritable traits, and I'm gonna depict those with different colors here. We have a population of living circles here, (laughs) and they could come in blue or maybe magenta. Maybe they come in another variation too, maybe there is yellow circles, and Natural Selection is all about which of these traits are most fit for the environment so that they can reproduce. So there might be something about being, say, blue, that allows those circles to reproduce faster, or to be less likely to be caught by predators, or to be able to stalk prey better. Even if they're only slightly more likely to reproduce, over time, over many generations, their numbers will increase and dominate, and the other numbers are less likely, or the other trait is less likely to survive, and so we will have this Natural Selection for that blue trait. So this is all about traits being the fittest traits. Now Genetic Drift is also change in heritable traits of a population over generations, but it's not about the traits that are most fit for an environment are the ones that necessarily survive. Genetic Drift is really about random. Random changes. Random changes, and a good example of that I have right over here that we got from, I'll give proper credit, this is from OpenStax College Biology, and this shows how Genetic Drift could happen. So right over here, I'm showing a very small population of 10 rabbits, and we have the gene for color, and we have two versions of that gene, or we could call them two alleles. You have the capital B version, and you have the lower case B, and capital B is dominant. This is kind of a very Mendelian example that we're showing here. And so if you have two of lower case genes, two of the white alleles, you're going to be white. If you have two of the brown alleles, the capital Bs, you're going to be brown, and if you're a heterozygote, you're still going to be brown. So as you can see here, there are several heterozygotes in this fairly small population. But if you just count the capital Bs versus the lower case Bs, you see that we have an equal amount of each. And so the frequency, if you were to pick a random allele from this population, you're just as likely to pick a capital B than a lower case B. Even though the phenotype, you see a lot more brown, but these six brown here have both the upper case B and the lower case B. Now let's say they're in a population where whether you are brown or whether you are white, it confers no advantage. There's no more likelihood of surviving and reproducing if you're brown than white, but just by chance, by pure random chance, the five bunnies on the top are the ones that are able to reproduce, and the five bunnies on the bottom are not the ones that are able to reproduce. And you might be saying hey, why did I pick those top five? I didn't pick them, I'm just giving an example. It could've been the bottom five. It could've been only these two, or the only two white ones were the ones that were able to reproduce. It's by pure random chance, or it could be because of traits that are unrelated to the alleles that we are talking about. But from the point of view of these alleles, it looks like random chance. And so in the next generation, those five rabbits reproduce and you could have a situation like this, and just by random chance, as you can see, the capital B allele frequency has increased from 50% of the alleles in the population to 70%. And then it could be another random chance, and I'm not saying this is necessarily going to happen. It could happen the other way. It could happen even though that first randomness happened, maybe now all of a sudden this white rabbit is able to reproduce a lot, but maybe not. Maybe these two brown rabbits that are homozygous for the dominant trait are able to reproduce, and one again it has nothing to do with fitness. And so they're able to reproduce, and then all of a sudden, the white allele is completely gone from the environment. And the reason why this happened isn't because the white allele somehow makes the bunnies less fit. In fact, it might have even conferred a little bit of an advantage. It might have been, from the environment that the bunnies are in point of view, it might have even been a better trait, but because of random chance, it disappears from the population. And the general idea with the Genetic Drift, so once again, just to compare, Natural Selection, you are selecting, or the environment is selecting traits that are more favorable for reproduction, while Genetic Drift is random changes. Random changes in reproduction of the population. Now, as you can imagine, I just gave an example with 10 bunnies, and what I just described is much more likely to happen with small populations. So much more likely. More likely with small populations. And we have videos on statistics on Khan Academy, but the likelihood of this happening with 10 bunnies versus the likelihood of what I just described happening with 10 million bunnies is very different. It's much more likely to happen with a small population. So a lot of the contexts of Genetic Drift are when people talk about small populations. In fact, many times Biologists are worried about small populations specifically because of Genetic Drift. For random reasons, you could have less diversity, less variation in your population, and even favorable traits could be selected for by random chance. There's two types of Genetic Drift that are often called out that cause extreme reductions in population, and significantly reduce the populations. One is called the Bottleneck Effect. Let me write this down. So the Bottle, Bottleneck, the Bottleneck Effect, and then the other is called the Founder Effect. Do that over here. The Founder, Founder Effect. They are both ideas where you have significant reduction in population for slightly different reasons. Bottleneck Effect is you have some major disaster or event that kills off a lot of the population, so only a little bit of the population is able to survive. And the reason why it's called Bottleneck is imagine if you had a bottle here. If you had a bottle here and, I dunno, inside of that bottle, you had marbles of different colors. So you have some yellow marbles, you have some magenta marbles, you have some, I don't know, blue marbles. These are the colors that I tend to be using. You have some blue marbles, so you have a lot of variation in your original population. But if you think about pouring them out of a bottle, maybe somehow there's some major disaster, and only two of these survive, or let's say only four of these survive, and so you could view that as, "Well, what are the marbles that are getting poured "out of the bottle?" It's really just a metaphor. Obviously, we're not putting populations of things in bottles. But after that disaster, only a handful survive, and they might not have any traits that are in any way more desirable or more fit for the environment than everything else, but they just by random chance, because of this disaster, they are the ones that survived. And so all of a sudden, you have a massive reduction not only in the population, but also in the variation in that population, and many alleles might have even disappeared, and so you have an extreme form of Genetic Drift actually occurring. Another example is Founder Effect, which is the same idea of a population becoming very small, but the Founder Effect isn't because of a natural disaster. Let's say you had a population. Once again, you have a lot of different alleles in that population. You have a lot of variation, you have a lot of variation in that population. So let me just keep coloring it. You have a lot of variation in this population, and let's say that, you know, they're all hanging out in their region, and maybe, you know, they are surrounded by mountains. I'm just making this up as I go, but let's say a couple of these blue characters were out walking one day, and they maybe get separated from the rest of their population. Maybe they discover a little undiscovered mountain pass, and they go settle a new population someplace. So that's why it's called the Founder Effect. These are the founders of a new population, and once again, by random chance, they just have a lot less variation. They're a smaller population and they happen to be disproportionately or all blue in this case, and so now this population is going to (mumbles) Just the process of this was Genetic Drift where many alleles will have disappeared because you have such a small population of blues here. And also because you have such a small population, you're likely to have even more Genetic Drift. So it's a really interesting thing to think about. Evolution and Natural Selection are often talked about hand in hand, but Natural Selection isn't the only mechanism of Evolution. You also have Genetic Drift, which is really about, not selecting for favorable traits, it is about randomness. |
88 | Biomolecules | Evolution Population Dynmaics | Inbreeding | So today, I want to talk to you about the effects of inbreeding and how it's not really the best for a population. So before we do that, let's review the concept of natural selection. And natural selection is the idea that a member of a population that has a special genetic trait that's advantageous is more likely to live to an age where they can reproduce and pass on that special trait to their offspring. And you should also remember that a population can get a lot out of having a big gene pool. And the bigger the gene pool, the more genetic diversity the population has, which allows the group to adapt to many different environmental changes. So what is inbreeding, exactly? Well, inbreeding is when people in a population will selectively have offspring with a certain smaller group within that larger population. And this can be for a bunch of reasons, like religion or culture, or maybe just because of preference. And when inbreeding occurs with nonhuman populations, it's almost always due to geographical barriers, where the greater population simply isn't accessible. Now, when people usually think of inbreeding, words like "incest" come to mind. But inbreeding really isn't limited to members of the same family having offspring together. Lots of small religious and cultural groups in the world have some people with common ancestors and are only distantly related. So you can see that the effects of inbreeding can exist without close relatives actually having children together. So why is inbreeding a problem in the first place? Well, let's look at an example. So TaySachs disease is an autosomal recessive disorder. And what that means is that people with no copies of the genes are unaffected by the disease. And I've drawn these people in blue. People with just one copy of the gene are not affected by the disease, but are carriers for the gene. And I've drawn these people in red. And people with two copies of the gene are affected by the disease. And I've drawn these guys in purple. So let's say we have someone who's a carrier for TaySachs. So he has just one copy of the gene. If we're looking at the general population, we can see that the odds of the person choosing a mate that's also a carrier for the disease are pretty low. And if he eventually has some kids, none of them will be affected by the disease, and only a few will even be carriers. It's likely that the copies of the gene will be so spread out among the population that it would be quite rare for two carriers to actually end up mating together. Now if we look at an inbred population where a bunch more people could be carriers for the disease, the chances of our guy choosing a mate that's also a carrier are a little higher. So more of his children will be carriers for the disease. But there's also a chance that some of his offspring may get two copies of the gene and actually be affected by the disease. Now, we just talked about an example with an autosomal recessive disorder. But maybe you're wondering how inbreeding affects autosomal dominant disorders. Well, let's look at Huntington's disease, which is autosomal dominant. And since this disease is autosomal dominant, if a person has no copies of the gene, they'll be unaffected by the gene. And I've drawn these people in blue once again. However, if a person has either one or two copies of the gene, then that person will be affected by the disease either way. And I've drawn both of these people in red. The key difference in this case is that no matter who the guy has children with, even if that guy just has one copy of the Huntington's gene, there's still a chance that there will be children affected by the disease. Now, of course, if our guy has children with someone who was also affected by the disease, then more of his children would be affected. But there's still a chance either way. Now, one of the other reasons why we're less concerned about inbreeding affecting autosomal dominant diseases is that carriers for dominant disorders are generally aware that they're affected and are well aware of the risks of them having diseased children. With recessive disorders, carriers usually don't have any symptoms at all. And they may not even know that they're carriers until they've had a diseased child. And this makes it much more important for people in inbred populations to seek genetic counseling so that they are aware of the risks of them having diseased children. So what did we learn? Well, first we learned that certain inbred populations can have many more individuals that may carry a diseased chromosome than the general population. But we also learned that this is mostly a concern with autosomal recessive diseases, since those generally go more unnoticed than dominant ones do. |
89 | Biomolecules | Evolution Population Dynmaics | Reproductive isolation | Have you ever wondered how we classify different organisms into different species? Well, before we look at that, let's go over the difference between asexual reproduction and sexual reproduction. In asexual reproduction, one organism, like a single bacterium, will divide into two daughter cells that are both genetically identical to the original cell. In sexual reproduction, two members of the same species will reproduce together in order to form genetically unique offspring. Now, in general, we say that organisms that reproduce asexually usually have low genetic diversity, whereas sexually reproducing species have high genetic diversity. So what is a species? Now, this can be a very difficult question to answer. For sexually reproducing organisms, we can say that two organisms, like this cat and this human, are members of different species. They're unable to have offspring together. However, for asexually reproducing organisms, like bacteria, protists, and archaea, it's a little more confusing. These species don't mate with other organisms. So we have a difficult time classifying them into different categories. And we call this the species problem. But in this video, we're going to spend time just looking at those sexually reproducing organisms. And these are separated into different species by different forms of what we call reproductive isolation. And this is the idea that there are many forces that stop two different organisms from having offspring together. And we can divide these forces into two separate categories, prezygotic forms and postzygotic forms. Prezygotic isolation refers to all the different forces that prevent two organisms from having offspring together that occur prior to the formation of a zygote. And remember that a zygote is a single cell that is made up of the genetic material of both organisms that have reproduced together. Postzygotic forms of isolation we'll get into a little bit later. So the first type of prezygotic isolation is temporal/habitat isolation. And temporal isolation refers to the fact that not all organisms mate at the same time. Some may mate at night, while others mate during the day. Some may mate in spring, while others mate in winter. If two organisms do not find mates at the same time, then they are temporally isolated. Habitat isolation refers to the place where the organisms mate. Some may prefer mating in the forest, while others prefer mating in the mountains. And if two organisms don't find mates in the same place, then they are also isolated. If time and place aren't a problem, then the next barrier is behavioral isolation, which refers to mate selection and how organisms go about attracting a mate. Now, not all organisms will attract a mate the same way. Perhaps one animal, like a bird, will attract a mate by singing a song, whereas this bunny rabbit may do a little dance to attract a mate. So we have behavioral isolation. And now we have mechanical isolation, which deals with the physical inability of two organisms to mate, even if they wanted to. Now, a great example of this is a huge animal like an elephant being unable to mate with a tiny mouse. If two organisms do mate successfully, they may still encounter gametic isolation, which is when fertilization between the two gametes to form a zygote is impossible. Now, once the zygote has been formed, we can move on and look at postzygotic forms of reproductive isolation. And the first form is zygote mortality. And this occurs when even if the two gametes from the two organisms can fuse successfully and form a zygote, that zygote would have a high mortality rate and be unable to develop into a mature offspring. Next we have hybrid inviability, which occurs when a zygote is able to grow into a mature offspring, but that offspring will have a high mortality rate and won't be able to grow into a mature adult. Finally, we have the last form of reproductive isolation, which is hybrid sterility. And this is when the offspring can grow into a mature adult. But that mature adult is not able to mate and have offspring of its own. So if two sexually reproducing organisms are not isolated by any of these barriers, then we can generally say that they are members of the same species. So what did we learn? Well, first we learned about the species problem and how classifying different organisms into different species can be quite difficult. We have a pretty good definition for sexually reproducing organisms, but not really for asexually reproducing organisms. And next we learned about reproductive isolation and how we can say that two sexually reproducing organisms are reproductively isolated if they are unable to freely produce fertile offspring together. |
90 | Biomolecules | Principles Of Bioenergetics | Gibbs free energy introduction | Our bodies are constantly active. Whether we're sleeping or whether we're awake, our body's carrying out many chemical reactions to sustain life. Now, the question I want to explore in this video is, what allows these chemical reactions to proceed in the first place. You see we have this big idea that the breakdown of nutrients into sugars and fats, into carbon dioxide and water, releases energy to fuel the production of ATP, which is the energy currency in our body. Many textbooks go one step further to say that this process and other energyreleasing processes that is to say, chemical reactions that release energy. Textbooks say that these types of reactions have something called a negative delta G value, or a negative Gibbsfree energy. In this video, we're going to talk about what the change in Gibbs free energy, or delta G as it's most commonly known is, and what the sign of this numerical value tells us about the reaction. Now, in order to understand delta G, we need to be talking about a specific chemical reaction, because delta G is quantity that's defined for a given reaction or a sum of reactions. So for the purposes of simplicity, let's say that we have some hypothetical reaction where A is turning into a product B. Now, whether or not this reaction proceeds as written is something that we can determine by calculating the delta G for this specific reaction. So just to phrase this again, the delta G, or change in Gibbsfree energy, reaction tells us very simply whether or not a reaction will occur. Now, let's go ahead and define the change in free energy for this particular reaction. Now as is implied by this delta sign, we're measuring a change. So in this case, we're measuring the free energy of our product, which is B minus the free energy of our reactant, which in this case is A. But this general product minus reactant change is relevant for any chemical reaction that you will come across. Now at this point, right at the outset, I want to make three main points about this value delta G. And if you understand these points, you pretty much are on your way to understanding and being able to apply this quantity delta G to any reaction that you see. Now, the first point I want to make has to do with units. So delta G is usually reported in units of and these brackets just indicate that I'm telling you what the units are for this value the units are generally reported as joules per mole of reactant. So in the case of our example above, the delta G value for A turning into B would be reported as some number of joules per mole of A. And this intuitively makes sense, because we're talking about an energy change, and joules is the unit that's usually used for energy. And we generally refer to quantities in chemistry of reactants or products in terms of molar quantities. Now, the second point I want to make is that the change in Gibbsfree energy is only concerned with the products and the reactants of a reaction not the pathway of the reaction itself. It's what chemists call a "state function." And this is a really important property of delta G that we take advantage of, especially in biochemistry, because it allows us to add the delta G value from multiple reactions that are taking place in an overall metabolic pathway. So to return to our example above, we had A turning into a product B. But what if the product B turned into another product C? If we wanted to calculate the overall Gibbsfree energy for A going to C, we could instead calculate the individual delta G for each step of the reaction that is A going to the product B, and B going to the product C. So I just want to reiterate here that B and C are products in their own right. They're not transition states. But what we're seeing here is that in some cases we may not be able to measure the change in Gibbsfree energy going from A to C directly. So instead, we can add together the individual change in Gibbsfree energy for each step, because remember Gibbsfree energy is a state function. And if we do that, we ultimately get the change in Gibbsfree energy for the overall reaction of A going to C. Now one fun way that I kind of remember the state function like quality of delta G, as well as some other variables in chemistry, is that my chemistry professor used to tell us that life is not a state function. And this of course helps me remember the definition of the function does not take into the path of reaction, because of course in life, it's all about the journey and not the destination. But in chemistry, sometimes it's the opposite. Now, the third point that I want to make is that delta G unlike temperature, for example, which can be readily measured in a lab for a particular situation, delta G is something that can be calculated but not measured. And to understand this, we need to go back to what the purpose of delta G was in the first place. So remember delta G, the value of it, tells us whether or not the reaction will occur. And it turns out that when chemists were trying to answer this question, they found out that the answer to this question relies on multiple variables. There's not just one thing that determines whether or not a reaction will occur. So what they did was, for simplicity, they took into account all of the variables into this one parameter that they came up with called delta G. And the way they did this was by creating an equation. So they said, the change in Gibbsfree energy is equal to the change in enthalpy, or heat content, of a particular reaction minus the temperature of the reaction times the change in entropy, or broadly speaking randomness, between products and reactants in a particular reaction. Therefore, as I mentioned before, we can go ahead and calculate one single value that takes into account all of the variables that affect the extent and degree to which a reaction will occur. And it turns out that we can actually measure the change in enthalpy, the temperature, and the change in entropy for a reaction, so that works out quite well. Now, at this point, you probably have a question of OK, I see that I have an equation to calculate delta G for a reaction, but what does this value that kind of pops out of this equation tell me about a reaction? So let's go ahead and go back to our hypothetical reaction of A going to B. Let's draw a diagram that will help us understand this reaction better. So I'm going to go ahead and draw a yaxis and an xaxis. On the yaxis will be the quantity free energy in units of joules, let's say. And on the xaxis will be the quantity of a reaction coordinate. And this is kind of an abstract parameter that simply is a way for us to kind of monitor the progress of a reaction over time. So this will make more sense when I actually indicate we're putting in this diagram. So let's say that our reactants A have a much higher free energy than the products of our reaction, which is B in this case. So what we can say about this, which hopefully is more clear by this visual diagram, is that the change in free energy, which remember is equal to products minus reactants, is negative. Or we say it's less than 0. On the other hand, let's say that we started off with reactant A that had a much lower free energy than the product B. Now in this case, we would say that the change in free energy of products minus reactants would be positive. Now, the key takeaway here is that for any chemical reaction that has a negative delta G value, we say that the reaction proceeds spontaneously. That is, it proceeds without an input of energy. So I'm just going to write spontaneous there. On the other hand, when a delta G value is positive, that is when the conversion of reactants to products requires a gain of energy, we say that it's a nonspontaneous reaction and cannot proceed unless there is an input of energy. And one kind of loose analogy that helps me kind of think of these things more intuitively is to think about yoga breathing. So imagine that you're taking a deep, deep breath in, and all of this breath that you have inside of your body makes you feel kind of unstable and wanting to burst. So I kind of think of that as starting off at a high free energy state. So let's say we're starting off with A. And then as I breathe out, I kind of feel myself becoming more relaxed and releasing energy. And that brings me to B, which has a lower free energy. And that of course, breathing out, is a spontaneous process. |
91 | Biomolecules | Principles Of Bioenergetics | An analogy for Gibbs free energy | So in this video I want to go ahead and talk more about the mathematical expression for delta G, or the change in Gibbs free energy, which I've went ahead and already written out here. So let's remind ourselves that this change in free energy is a quantity that is equal to the change in enthalpy, or heat content for a reaction minus the temperature at which the reaction is run times the change in entropy or, broadly speaking, disorder in going from reactants to products for a particular chemical reaction. OK so that was kind of a mouthful, right? So my ultimate goal in making this video is to ultimately present to you an analogy that I hope will give you a better intuitive feel for how the terms of enthalpy and entropy contribute to the overall sign of delta G. Because remember it is the sign of delta G, that is whether it's negative or positive, that tells us whether or not a reaction is spontaneous or nonspontaneous. But before talking about analogies let's go ahead and get a better handle on enthalpy and entropy from more of a chemistry and mathematical lens. So recall that enthalpy is really a good proxy for the change in bond energy that occurs during a reaction, that is to say whether or not energy was released or absorbed during a bond rearrangement. And because all systems want to achieve their lowest possible energy, if we're talking about enthalpy in isolation from entropy, let's say, generally speaking we might intuitively say that having a negative delta H, which describes a release of energy remember going from reactants to products, would be more favorable than a positive delta H value. On the other hand, the second law of thermodynamics says that all systems tend toward disorder and since entropy, or the chain in entropy, is a measure of whether something is getting more disordered or less disordered. In isolation, if we're just talking about entropy, we might intuitively say that having a positive delta S value, that is to say describing an increase in entropy from going to products from reactants, would be more favorable than a negative delta S value. Now mathematically our equation supports our hypothesis, right? A negative number minus a positive number will always be negative. That is to say regardless of what the temperature is, the delta G value will always be negative, the reaction will always be spontaneous. But since delta H is not always negative, and since delta S is not always positive for all reactions, what will the delta G be for other combinations of delta H and delta S? So to explore this further I thought we could go ahead and create a table like such to write out essentially the different signs that delta H and delta S could take on, and determine for each of these combinations whether the delta G will be positive or negative. So we've already gone through one. We've said that when delta H is negative, and delta S is positive, delta G will be negative. But of course there are other combinations too, delta H could be positive, and delta S could be negative, or both delta H and delta S could be positive, or delta H and delta S could both be negative. So let's go ahead and take these signs and plug and chug into our equation above. So if we have a positive number and we're subtracting a negative number, we are always going to get a positive number. So for all situations in which delta H is positive and delta S is negative we will have a positive delta G value. This makes sense, right? Because it's essentially the opposite of the previous situation. But what about the case in which you have a positive number minus another positive number? Well, you might say it really depends on the relative magnitude of these two numbers, right? Because if you have a big positive number minus a smaller positive number you would get a positive number. But if you had a small positive number minus a bigger positive number you would get a negative number. So ultimately it really depends, delta G can either be positive or negative. And this value of temperature plays a pretty big role in kind of tipping the scale towards one direction or another. Because if it's really high, for example, our second term might be more positive, and therefore this overall value might be more negative. But if it's not very high and delta H is really, really large, well, perhaps then delta G would be positive. So finally let's look at the situation where we have a negative number in our last row minus another negative number. Well, you might say again this really depends, right? If you have a small negative number and you're adding a very large positive quantity to it, it could be positive. But if it's a smaller number that you're adding on to a negative number, it could still be negative. So again this really depends, and again temperature plays a big role in tipping the scale one direction or the other. Now for the analogy that I want to present, I really want to focus on these last two rows here in which temperature plays a big role in determining whether or not a reaction is spontaneous or not. So how can we understand this tradeoff between delta H and delta S at high or low temperatures? So I'm going to go ahead and scroll down and I'm going to present an analogy that I hope isn't too corny. But hopefully it's useful, and you might even consider thinking of your own analogy to help you understand this tradeoff between entropy and enthalpy. So imagine you are a chemistry student, and for spring break you decide to go with your friends somewhere really tropical, where there are a bunch of palm trees and great beaches. And one day you and your friends go ahead out to one of the beaches and you and a couple of friends set out a large beach towel and decide to sunbathe on the beach, while some of your other friends decide to go out into the ocean and swim. Now of course being a chemistry nerd you describe this phenomenon in terms of a chemical reaction, that is to say a reaction in going from lying down on a beach towel to swimming in the water, or the reverse that is going from the water to lying on a beach towel. Now at this point you begin thinking back to your equation for delta G, which remember is equal to delta H minus the temperature times delta S. And you begin to realize that the forward reaction that is going from a beach towel to the ocean is more favorable, that is to say if more of your friends are in the water when the sun is really at its peak, and everyone really wants to go to the water to cool off. On the other hand, the reverse reaction that is going from the water to the beach towel is more favorable when the sun is less intense, when it's less scorching hot outside and it's easier to relax on the beach. In other words, you conclude the sun is figuratively, or perhaps even literally here, analogous to the temperature variable in our equation. Depending on how hot the sun is ultimately determines whether or not it is more spontaneous to go into the water or come out of the water. Now since you happen to also be an English major and love metaphors, you go ahead and think up a metaphor for both the enthalpy and entropy contributions in your hypothetical chemical reaction. Enthalpy, you decide, you can think about in terms of the amount of energy that you're expending whether you're on a beach towel or in the water. So we all know that lying on a beach towel is super relaxing and very peaceful, and requires little to no energy and you're in a very, very stable place. But on the other hand, when you're swimming in the water it requires a lot of energy be expended because you're of course swimming and throwing around a beach ball. On the other hand I'm going to go ahead and scroll down here lying on a beach towel can kind of get boring after a while, right? But being in the water is lots of fun, and there's really never a boring moment. And this interplay between fun and boring is something that you can kind of think of as your entropy variable. Now in an ideal world you'd want to be both relaxed and having fun. And in chemistry language that's really another way of saying that you'd want to have a negative delta H value and a positive delta S value. But of course in this particular setup, these things are mutually exclusive. So what wins? Ultimately, in the end, you conclude that when the sun is out this fun term, or what we're kind of calling entropy wins, and the forward reaction is spontaneous. But when the sun isn't as hot outside this relaxation, or enthalpy term, wins and the reverse reaction is spontaneous. And with this mighty conclusion that you have come up with on this relaxing day at the beach you go home and reminisce about how chemistry and life are far more similar than you once thought. |
92 | Biomolecules | Principles Of Bioenergetics | Heat transfer | Imagine that you are holding in your hands a glass of water. We're going to say that the glass of water is at room temperature, so that's about 20 degrees Celsius, and you are at body temperature, which is about 37 degrees Celsius. Now intuitively, you know that after a while, your hand starts to feel kind of cold, right? So what's going on here? In the physical sciences, we say that what's going on here is called a heat transfer. And the idea is that because we have two different things at different temperatures, specifically because our water's at a lower temperature, energy in the form of heat is going to travel from our body to the water, which means that our body is losing energy, and the cup of water here is gaining that energy. So we are becoming cold, and the cup of water is warming up. Now when I was first learning about heat transfer, I didn't quite understand the difference between heat and temperature, so I just want to go ahead and briefly discuss what the difference is. Heat is the amount of energy that's transferred due to a change in temperature. So I'm going to go ahead and write that out here. Remember that whenever you see a gradient, whether it's a gradient of pressure, or concentration, or in this case, a gradient of temperature, it always means that there is some type of potential energy that's stored up. And since the system wants to achieve its lowest energy possible, if there is no opposing force, the gradient wants to disappear, so this is the basis behind heat transfer. Of course, what sets up this gradient is temperature, and temperature is an absolute quantity. Specifically, it's defined as the average kinetic energy of molecules in whatever we are measuring the temperature of. So to summarize, the point I really want to underscore here is that while temperature is an absolute measure of energy, there is really no such thing as an absolute heat content. Heat should always be described as heat transfer, because it is measuring the amount of heat that is either lost or gained. It's also important to emphasize that heat transfer occurs between a system and its surroundings, which I'm going to abbreviate as surr. So to understand this, let's go ahead and revisit our example above. Now, what's really important to determine here is the direction of heat transfer. Now as I talked about before, we can't really attribute an absolute value of heat content to either the system or the surroundings, but what we can say is, what direction does this heat transfer occur in? What's getting hotter, essentially, and what's getting colder? So the way that physical scientists kind of denote heat transfer is with the lowercase letter q. And so in the case of our system, which is our hand which is becoming colder, the way that this would be denoted is with a minus q, because heat energy is being lost from the system. On the other hand, what you would find after a while if you kept holding this glass of water and then set it down on the counter and measure the temperature is that the temperature of the water would have increased. And essentially, what is going on here is that the energy lost from the system is being absorbed by the surrounding. And so in this case, because heat energy is being gained, this would be denoted as plus q. To state this phenomenon more generally, it's fair to say that the heat that is either lost or gained by the system is equal and opposite in magnitude to the heat that's either lost or absorbed by the surroundings. |
93 | Biomolecules | Principles Of Bioenergetics | Enthalpy | So we've been talking kind of at a very macro level about heat transfer by using an example of heat being transferred between our hand and a glass of water. So now let's go more of a micro level. Let's talk about heat transfer and chemical reactions. So when we're talking about heat transfer and chemical reactions we're talking about the term called enthalpy. Now it turns out that if we think about chemical reactions as our system, so go ahead and write that here, chemical reactions can either release or absorb energy that can change the temperature of its surroundings. Which we generally think of when we're talking about chemical reactions as the solution that the chemical reaction is taking place in, and of course if the chemical reaction is taking place in our body the surrounding is really just our body, which you can think of really as just a big bag of water. So a question might be on your mind. Why did chemists come up with this fancy term enthalpy to describe the heat transfer of chemical reactions? It turns out that enthalpy is a very useful quantity to calculate for many chemical reactions because not only does it tell us something about heat transfer, but it also is a component of Gibbs free energy, which is an important parameter that chemists use to determine whether or not a reaction will take place or not. Generally speaking, I think it's OK to conceptualize with some simplification that enthalpy is essentially just a fancy term to describe heat transfer for chemical reactions. And because we can never define an absolute quantity of heat, because remember heat is the amount of energy transferred, it's not an absolute term, enthalpy is always referred to as a change in enthalpy, and oftentimes it's written as delta H. And specifically this change in enthalpy describes the change in heat energy. That is whether heat was lost or gained from the perspective of the system. So even thought that there is this kind of important interplay, this conservation of energy between the system and the surroundings, this term enthalpy is really just telling us what's happening from the perspective of the system. And I think this will make more sense as I give you an example below, but before I do that I just want to note what the units of enthalpy are. And the units of enthalpy are joules per mole of reactant in the chemical reaction. And this of course makes sense because joules is a unit of energy, and we're talking about heat, which is a form of energy. And it's notable to know that having it per mole allows us to take the amount of whatever is reacting into account. This is something that we can't really take into account if we just, say, measure the change in temperature that occurred over the course of a reaction. All right, so now let's go ahead and take a look at an example. So I'm going to go ahead and scroll down. Let's go ahead and use an example that you will be fairly familiar with. So when our bodies get super hot to cool ourselves down our bodies essentially evaporate water from the surface of our body to help cool ourselves down. And this is really just a fancy way of saying that we start sweating. Right? Now the chemical reaction for sweating we can really think about as just essentially the evaporation of water. So that is to say taking water from the liquid phase and turning it into water vapor, or water in the gaseous phase. The change in enthalpy for this particular reaction, and note that I'm saying for this particular reaction, for our system, is defined as the enthalpy of our product, which is our water in its gaseous form, minus the enthalpy of our reactant, which is water in its liquid form. This of course is a way to conceptualize enthalpy, but remember it's really actually not possible to measure an absolute quantity of enthalpy for anything. We can only ever measure this change in enthalpy, which involves monitoring the change in temperature of the surroundings during the course of a reaction as well as some other considerations that we're not going to go into in this video. But just to note that it's really the change in enthalpy that we're measuring and not these absolute quantities, even though technically this is how we're conceptualizing enthalpy. So the key idea here about this process of evaporation is that it requires energy to occur. Just think about boiling water. In order to get water into its gaseous phase you need to heat it up. So the fact that we're adding energy to our system should tell you something about what this sign of this change in enthalpy should be. The products are essentially gaining more energy in the form of heat than the reactants. So we say that in a process that absorbs heat the change in enthalpy is greater than 0. In other words, the change in enthalpy is positive. And whenever a reaction has a positive change in enthalpy, which by definition means that heat is being absorbed, we call it an endothermic reaction. And the way that I like to remember this is that endo is I think a Latin prefix for within. So heat is going in to our system, which is our chemical reaction. So you're probably wondering at this point, well, if we're absorbing heat, how is this connected to us cooling off? Well, this is kind of the important point that I alluded to before. This change in enthalpy only tells us what's going on with our system, which of course is our chemical reaction, but our body in this case is our surrounding. So we can use this relationship above that tells us that the heat lost or gained by the system is equal and opposite to the heat lost or gained by the surroundings. So in this case we know that our system is absorbing heat, which means that our surroundings, our body, must be losing heat. And that's how we cool down. Now you can also imagine that we have chemical reactions in which the change in enthalpy is less than 0, and we call these types of reactions exothermic reactions. And the way I kind of think about these is that ex, this ex term is kind of a Latin prefix for out of. So heat essentially is going out of the system instead of being absorbed. One example of a very prevalent reaction in our bodies that is exothermic is the hydrolysis, or reaction with water, of ATP, which is of course the energy currency of our cells. So ATP reacts with water, and it loses a phosphate group to become ADP and a free phosphate group. And this reaction has a negative change in enthalpy, or in other words it releases heat. The fast that this reaction is exothermic is physiologically significant for many reasons. But one of those reasons is when we're talking about shivering. So we all know that we shiver when we're cold. And the reason we do that is because we want to warm ourselves up. And that heat energy is indirectly tied to this hydrolysis of ATP which releases heat and allows our muscles to contract to warm us up. So just to wrap things up here, I think the key takeaway is that enthalpy describes heat transfers for chemical reactions, and notably it's from the perspective of the chemical reaction, not the surroundings. And so chemical reactions can either lose heat, in which case they are classified as exothermic reactions and having a negative enthalpy, or they require an input of heat, and they're classified as endothermic, and have a positive change in enthalpy. |
94 | Biomolecules | Principles Of Bioenergetics | Le Chatelier's principle | Let's say we had the reaction molecule A plus molecule B is in dynamic equilibrium with molecules C plus D. Which just means that the rate of the forward reaction is going at the same rate as the backward reaction, or the reverse reaction. There will be some equilibrium concentrations of A, B, C, and D, and we can figure out what the equilibrium constant is if we want. And I'll say it again. I've said it like four times so far. The fact that the forward reaction rate is the same as the backward reaction rate doesn't mean that all of the concentrations are the same. The concentrations themselves of each of the molecules could be very different. They're just not changing anymore because the forward and backward rates are the same. Now, given this, given that we're at equilibrium now, what's going to happen if I add more A to the system? So remember, it was in equilibrium. The concentrations were constant. But now all of a sudden, I'm adding more A to the system. So now, the odds of an A and a B particle, even though I'm not adding any more B molecule to the system, the odds are slightly higher that an A and a B are going to collide in just the right way, so the forward reaction is going to be more likely. So if we add more A to the system, you're going to have more A's. They're going to bump with more B's, so the B's are actually going to go down a little bit, right? Because more B's are going to be consumed. And even more important, the C's and D's are going to definitely be increased. And the way it would really happen, you would add more A. Those A's would bump into some more B's, and so this forward reaction, all of a sudden, it's rate would go faster than this backward reaction. So the reaction would go in that direction. Then you would have more C's and D's and maybe some of those are more likely to bump and go back in this direction. Eventually, you would reach a new equilibrium. But the bottom line is you'll be left with more A, a little bit less B because you didn't add more B. So more B's going to be used to consume with those extra A's you just added. And then those are going to produce more C's and D's in equilibrium. And you can imagine, if you added more A and more B, let's say if you added more B as well, then the reaction is going to go in the forward direction even more. I don't think this is an amazing insight of the world. I think this is kind of obvious, that if you stress this reaction by adding more on this side, that naturally it's going to move in the direction that relieves the stress. So if you add more A, you're going to have more A's bumping with B's and go in that direction and maybe consume a little bit more B's. If you add more of both, the whole thing's going to go in that direction. Likewise let me rewrite the reaction. I'll do it in a different color. A plus B, dynamic equilibrium, C plus D. If I add more C I think you get the point here what's going to happen? Well, that's going to drive A and B up, and it's maybe going to consume a little bit extra D. And then if you added more C and D, then, of course, it's going to produce a lot more A and B. And this idea, it seems pretty common sense, but there's a fancy name for it, and it's called Le let me put a capital L Chatelier's principle. If you've watched enough of these videos, you know I have to be careful with my spelling. And all it says is that when you stress a reaction that's in equilibrium, the reaction will favor the side or one side of the reaction to relieve that stress. When they say stress the reaction, that's like adding more A, so the reaction's going to move towards the forward direction to relieve the stress the quote, unquote stress of that more A. I mean, that's not stress in its traditional way of thinking about stress, but that is a kind of stress. You're somehow changing it relative to it. It was nice and comfortable before in a nice, stable environment. So given Le Chatelier's principle, let's think of some other situations. Let's say if I had A plus B plus some heat, and that produces some C plus D. And maybe it produces some E as well. So if I were to add heat to this system, what would happen? So in order for the reaction to progress in the forward direction, you need heat. The more heat you have, the more likely you're going to progress in the forward direction. So Le Chatelier's principle will say we're stressing this reaction by adding heat, so the reaction will favor the direction that relieves that stressor. And so to relieve that stressor, you have more of this input, so you're going to consume more A. So the stable concentration of A once we reach equilibrium will go down. B will go down because they're going to be consumed more. The forward reaction is happening more. And then C, D, and E would go up. Now, if you did the opposite. Let me erase what I just did. Let's say instead of adding heat, you were to take away heat. So let's say you were to take away heat. Let me make sure my cursor's right. So if you took heat away from the reaction, what will be favored? Well, then you're going to be favoring it in the other direction because there'll be less heat here. I mean, all of this is together. There'll be less heat for this reaction to occur, so this rate will start dominating this rate over here, right? If you take away heat, the rate of this reaction will slow down, this one will be bigger, and so you'll have more movement of concentration in that direction, or the reverse reaction will be favored. Now, let's think of another stressor pressure. Now, imagine that we had we mentioned the Haber process before, and this is the reaction for the Haber process. Nitrogen gas plus 3 moles of hydrogen gas in equilibrium with 2 moles of ammonia gas. Now, what's going to happen if I apply pressure to this system? I'm going to apply pressure. So if you think about what happens with pressure, everything all of a sudden is getting squeezed, although the volume isn't necessarily decreasing, but something is somehow making all the molecules want to be or forcing them to be closer together. Now, when things are getting closer together, the stress of the pressure could be relieved if we end up with fewer molecules. Think about it this way. PV is equal to nRT. We learned this multiple times, right? And let's say we could write P is equal to nRT/V. Now, if we increase the pressure, how can we relieve that? Remember, Le Chatelier's principle says that whatever's going to happen is going to relieve the stressor. The reaction is going to go in the direction that it relieves it. Well, if we lower the number of molecules, then that will relieve the pressure, right? You'll have fewer things bouncing against each other. So if we lower the number of molecules where you can kind of view it I mean, I shouldn't have written it this way, because it's not quite an equation, but I want you to think of it that way. Let me erase this. This probably wasn't the best intuition. If I have a container nope, too shocking. If I nope, same thing. If I have a container and I'm applying pressure to it, and in one option I could have 2 molecules let's say I could have 4 molecules in some volume. And in another situation, let's say they get merged and I only have 2 molecules, right? In either of these, the reaction can go between these, these 4 could merge to make 2 molecules. Actually, let me use this example up here. Let's say this nitrogen molecule is this blue one here. Actually, let me do it in a more different color. This brown one right here, it can merge with 3 hydrogen. It could produce this. So this is another way of writing this reaction, maybe in a more visual way. Now, if I'm applying pressure, if I'm applying pressure to this system, so pressure I just imagine is kind of more force per area from every direction, which of these situations is more likely to relieve the situation? Well, the situation where we have fewer molecules bumping around because it's easier to kind of apply or I guess squeeze them together than when you have more molecules bumping around. I'm doing this very hand wavy, but I think it gives you the intuition. So if you apply pressure to the system, if pressure goes up, you're applying this doesn't mean the pressure goes down. This means pressure is applying to the system. But the pressure is going up, what side of the reaction is going to be favored? The reaction's going to be favoring the side of that has fewer molecules. And this side has 2 molecules, although they'll be bigger molecules obviously, because it's not like we're losing mass in one direction or the other, as opposed to this situation where we have 4 molecules, right? 1 mole of nitrogen gas and 3 moles of hydrogen. And just to bring this all back to the whole idea that we saw earlier with the kinetic equilibrium, let's just imagine a reaction like this. And to show that it works with Le Chatelier's principle is consistent with everything we've learned with equilibrium constants. So let's say we had the reaction 2 moles, or the coefficient of two, 2 A's in the gaseous form plus B in the gaseous form is in equilibrium with C in the gaseous form. And let's say initially where our first equilibrium, our concentration of A is 2 molar, or our molarity is 2, our concentration of B is 6 molar concentration, and then our concentration of C is 8 molar. So what's the equilibrium constant here? The equilibrium constant here is the product, concentration of C, that's 8 molar divided by 2 molar squared, because of this, 2 squared times 6. Which is equal to 8/24, which is equal to 1/3. Now, let's say we were to add more A, and I'm not going to say exactly how much. We could actually get quite complicated with the mathematics, but let's say after adding more A, we have a new concentration. Now, let's say our concentration of A is 3 molar. You might say, hey, Sal, didn't you add 1 molar? No. I actually added probably more than 1 molar. What happens is, whatever I added, that'll push the reaction towards the right direction, or towards the forward direction, and so some of it will get consumed and go in that direction, but whatever's left over is here. So I might have added more than 1 molar concentration to this system. But whatever was extra beyond the 1 was consumed, and I'm just left with this equilibrium concentration of 3. So I didn't necessarily add 1. I could've added more than that. And let's say that our new equilibrium for C is 12 molar, which is consistent with what we say. We should be producing if we add some A, then our concentration of C should go up, and the intuition is that the concentration of B should go down a little bit, because a little bit more B is going to be consumed, because it's going to be colliding with or it's more likely to collide with more A particles or A molecules. So let's see what B's new concentration is. So remember, the equilibrium constant stays constant. So our equilibrium constant is now going to be equal to the concentration of C, right? That was the reaction. So it's 12 molar whoops I don't have to write the units here divided by our new concentration of A, that's 3. But remember the reaction. The coefficient on A is 2. So it's 3 squared times the new concentration for B, right? There's no coefficient here so I don't have to worry about any exponents. And let's just solve this. So you get 1/3 is equal to 12 over 9B. So if we just crossmultiply, we get 9 times the concentration of B is equal to 3 times 12, which is 36. And so divide both sides by 9. The new concentration of B is 4, or 4 molar. So B is equal to 4 molar. So that makes sense. We added some more A to the reaction. So we started with 2 molar of A, 6 molar of B, 8 molar of C. When we added more A, at the end, we added a bunch. It went in that direction, maybe it went back and forth a little bit, but it stabilized at 3 molar of A, 12 molar of C, so C went up, so that definitely went up. And notice, our stable equilibrium concentration of B actually went down, and this is consistent with what we were saying, that the reaction moves in that direction, more C gets produced, a little bit of B gets consumed. So anyway, hopefully you're fairly comfortable now with the whole notion of stressing a reaction and Le Chatelier's principle. |
95 | Biomolecules | Principles Of Bioenergetics | Thermodynamics vs kinetics | In this video, I want to go ahead and talk about thermodynamics and kinetics. I remember that when I was first learning about these two things in the context of chemical reactions, I used to hear phrases like, oh, this chemical reaction let's say hypothetically A going to B is kinetically favorable, so it must proceed. But then the next day when I go to class, I'd hear another statement that said, oh, well this reaction A going to B is thermodynamically favorable, so it must proceed. So in this video, I want to go ahead and clarify these two statements for you and really understand what it means to thermodynamically favored and/or kinetically favored. So to get it started, I've already drawn two plots so that we can plot out the free energy change that occurs for the forward reaction we'll do that on the left side and the reverse reaction, which we'll do on the right side. So before we get into that, let's go ahead and label our axes. Our yaxis in both cases will be measuring free energy, which is in units of joules. Then our xaxis will be an abstract dimension called the reaction coordinate, which essentially allows us to monitor the progress of a reaction. So now I'm going to go ahead and say that the forward reaction we'll draw out in this teal color. And the reverse reaction we'll go ahead and draw out in this pink color. So let's go ahead and start with the forward reaction. So I'm just going to go ahead and say that the forward reaction has a negative delta G value. So remember that means it's spontaneous, and visually that means that our reactants start off at a higher free energy than our products. Now, this of course means that the reverse reaction, which will have the same magnitude of delta G, that is the free energy change, will be at the same numerical value. But of course, since the reaction is going the opposite direction, the sign of delta G will be now positive. And visually, we're saying essentially that our reactant, which in this case is B, starts our at a lower free energy than our product, which is A. Now thus far in drawing this free energy diagram, we've just been talking about thermodynamics. But it turns out that there is also a kinetic energy barrier for the conversion of reactants to products, regardless of whether the reaction is spontaneous or nonspontaneous. This kinetic barrier of energy is referred to as the free energy of activation, or simply activation energy. So I'm going to go ahead and put in parentheses E sub A, which we'll say stands for activation energy. And remember that delta G of course is talking about thermodynamics. With that said, let's go ahead and add this kinetic energy barrier to our diagrams. And we can do this by understanding that the activation energy is defined as the amount of energy that is required to form a highenergy intermediate during the course of the reactions. In other words, in our hypothetical reaction of A going to B, it proceeds through an intermediate, that is a highenergy chemical product that won't last very long, but is important in the conversion of A to B or vice versa. So in our diagrams here, we can go ahead and indicate that there is some intermediate, so in between our reactants and our products, that is at much higher energy than everything else. And we can go ahead and then connect the dots. So go ahead and essentially draw a line from reactants to products that includes our highenergy intermediate. And this ultimately allows us to see the presence of the activation energy as well as the change in free energy. So let's go ahead and actually label these things. So on the left side here, remember our change in free energy, which is looking at our reactants compared to our products we're ignoring this little bump. The change in free energy, which I'm going to indicate with the green line, extends between these two points. And the same on the right side, just again extending between the start and endpoints of our reactions. So I'll go ahead and indicate that this green line in both cases refers to our delta G values. And in a different color, let's say red, I'm going to go ahead and indicate the activation energy, which takes into account the change in energy between the highenergy intermediate and the reactants. So in the case on the left, that change is indicated here with red. And on the right side, the change between the intermediate and the reactant is a bit longer, so we'll go ahead and indicate that here. Now, activation energy is an important quantity to take into account, because in order for molecules to react, they must have enough energy to overcome this activation energy barrier. Essentially, in the case of a spontaneous reaction for example, I think of it like the energy one needs to get a ball to start rolling down a hill. We all know that gravity will make a ball roll down a hill, which is like a negative delta G value, it's telling us that the reaction is very thermodynamically favorable. But we need to sometimes give the ball push in order for the reaction to occur. And so that's kind of this little help that it needs to go over before it can actually proceed. For a nonspontaneous reaction, the idea is essentially the same. We still need to have some activation energy. But in addition, because it requires an input of energy, we can think about it as rolling ball up a hill instead of down a hill. Now in general, the idea is that the lower this free energy change, the faster a reaction will occur. And remember I'm saying faster, so I'm talking about kinetics. I'm talking about the rate of a reaction. So just to write that out, the activation energy, the smaller it is, the faster the reaction will proceed. Now in biochemistry in particular, it's really important to distinguish between these two terms of thermodynamics and kinetics, which we've drawn out in our diagrams as the change in delta G over the change in activation energy. Because many biochemical reactions in our body are kinetically unfavorable, that is to say they have a very high energy of activation even if they are thermodynamically favorable. This is why our bodies have enzymes, which essentially lower the activation energy of a reaction. So I went ahead and drew a dotted white line that's a little bit lower, so you can see that when an enzyme is present, the height of the barrier has decreased. And if it's decreased, the reaction will proceed faster. Now, there's one analogy that my chemistry professor used to tell us all the time that really helped me understand the interplay between kinetics and thermodynamics as they apply to whether or not a reaction will occur. So I'm going to go ahead and scroll down so we can briefly talk about this analogy, which is I think a fun way to think about all of this. So let's say you went to a dating website, because you were looking for your perfect match. And this dating website told you that your perfect match lived halfway across the globe. And this is such a perfect match, and they have all these algorithms. And if this match were a chemical equation, we would say that you had a very, very negative delta G value. That means you would be very, very spontaneous. But you live halfway around the world, so in terms of chemistry language, we might say that you are kinetically limited. That is to say, you don't have any way of actually travelling halfway across the world to meet your special someone. So we might say that you have a very high activation energy. So from this discussion, perhaps the biggest takeaway is that neither kinetics nor thermodynamics solely determines whether a reaction will proceed. It's important to take both into account. Now one of the applications of this, in biochemistry especially, is remember that enzymes lower the activation energy of a reaction. So we can think of enzymes as light switches. They can regulate whether a reaction will proceed or won't proceed. But, of course, a light switch only works if the light bulb itself is working. And so the metaphor for the working light bulb is saying that a reaction has a negative delta G value. Or if light bulb is not working, we're saying that the delta G value is positive. That is, it requires a new battery or energy for the reaction to proceed. |
96 | Biomolecules | Overview Metabolism | Overview of metabolism: Anabolism and catabolism | Textbooks define metabolism, a topic in biochemistry, as a series of chemical reactions that take place inside of our bodies to sustain life. Now, this is a pretty broad definition of metabolism. So in this video, I really want to break this definition down to a more workable understanding of what metabolism really is. So first, I'm going to introduce another arrow in this diagram, like this, and say that really, the requirements of life, let's say in a human being, such as maintaining a constant internal temperature, reproducing, growing, and all that jazz, all of that ultimately boils down to the body's ability to utilize four essential biomolecules. And these four essential biomolecules, or as they're sometimes known as macromolecules, are proteins, fats, carbohydrates, or carbs, and nucleic acids, like DNA and RNA. And ultimately, all of these biomolecules perform different lifesustaining reactions inside of all of the cells in our body to ultimately promote life. So as you can see, we've already begun to break down this definition of metabolism. Essentially what we're saying here is that metabolism is really the study of how we're able to obtain these important biomolecules to sustain life. So how do we obtain these biomolecules? Now, a simple answer to this question is, of course, that we eat food to obtain all of these important biomolecules. But there is an important word of caution here, which is that since most food comes from living organisms like plants and animals, these plants and animals also contain an array of proteins, fats, carbohydrates, nucleic acids, but not necessarily in the same flavor or configuration that our bodies would prefer. So what do our bodies do instead? Well, in our bodies, we go ahead and eat the food. That's a very large head there, but you get the idea. And in our bodies, we break down this food through a process called digestion into the component parts of all of these biomolecules. So what do I mean by component parts? Well, the smallest subunit of proteins is called an amino acid. And our body breaks down all the different types of proteins that we digest into individual amino acids. And the same pattern continues for the rest of the biomolecules. So in the case of fats, we're talking about fatty acids, which are the smallest subunits of fats. And then for carbohydrates, which are long chains of sugars. One of the most common subunits of carbohydrates that our body loves is called glucose. So I'll go ahead and write that here, since you'll be seeing it a lot in the discussion of metabolism. And then finally, for nucleic acids were talking about nucleotides. So at this point, you're probably thinking, well, OK. I understand that our body can't use the same macromolecules found in food because maybe they're not in the right configuration. But how does breaking them down do anything for us? Now the key here is to recognize that in our body there is actually a delicate balance going on between the processes of breaking down molecules, such as in the process of digestion, and then taking these products and building them back up. So essentially, you can see all of these subunits, or monomers, as LEGO pieces that we're essentially reconstructing to build the right configurations of proteins, fats, carbs, and nucleic acids that our body needs. So that's really the key idea here, which is that metabolism is a balance between breaking things down and building them back up in our body so that we can customize, so to say, what type of macromolecules that we create. And just to throw in some vocab words, biochemists call the process of breaking down molecules in our body catabolism. And similar sounding word called anabolism is used to describe the process of building molecules back up. And the way I like to remember this is looking at the first letter of each of these words, I think of C, I think of cutting molecules up into tiny pieces, so breaking them down. And then for anabolism, A, I think of as like the apex of a building, for example. So we're building something up. Now this seems all fine and elegant, but there's one more issue that we need to contend with, which is a consequence of having to balance breaking things down and building them back up. And that is that this process of building molecules back up requires energy. Which I'm kind of indicating here by these yellow lightening bolt stars. So the question I want to answer in this last part of the video is where does this energy come from? Now, the answer to this question is that, well, we also get this energy by eating food. So how does that work? So first, recall that the energy currency of the cell and I'm going to go ahead and erase this just to give us some more space. The energy currency of our bodies is a molecule called ATP, or adenosine triphosphate. And this high energy molecule, as it's often referred to, when it is broken down into ADP, so it loses a phosphate group, it releases usable chemical energy that can fuel energy requiring processes in our body, such as the building up process of anabolism. Now, in order for this process to continue nonstop in our bodies, ADP must be regenerated into ATP. And that is where food comes in. So remember that we digest our food into all of these subunits. And some of these subunits, such as glucose and fatty acids mainly, but occasionally amino acids I'm going to put that in parentheses can essentially be used as fuels in our body. So just like wood, for example, is a fuel for a burning fire, which produces heat, these fuels in our body can essentially be broken down even further to produce the energy that's necessary to convert ADP back into ATP and thus allowing this cycle to continue. And just to throw in another vocabulary word that you'll probably see, this process of taking these fuels, which I've indicated with this asterisk, and breaking them down into usable energy is a process that's referred to as cellular respiration. And recall that because cellular respiration involves breaking down things even further, it's also a catabolic process. So it falls under this category of catabolism. And just to tie everything here together at the end, notice here that another way to interpret this cycling between ATP and ADP is to say that catabolism fuels anabolism. So what do I mean by this? Well, essentially, catabolism, such as the process of breaking things down and extracting energy through processes of cellular respiration, is coupled with this process of building things back up. And so in essence, one relies on the other. And as you can probably guess, these processes are really tightly regulated in our bodies. Because obviously you wouldn't want to be breaking down something while you're building something back up. And in fact, just to give you a preview forward, catabolism and anabolism are often regulated, so controlled, through the use of hormones. So I'm going to write here that hormones are a form of regulation, and tell the body whether it should be in a catabolic or anabolic state. |
97 | Biomolecules | Overview Metabolism | ATP: Adenosine triphosphate | Sal: ATP or adenosine triposphate is often referred to as the currency of energy, or the energy store, adenosine, the energy store in biological systems. What I want to do in this video is get a better appreciation of why that is. Adenosine triposphate. At first this seems like a fairly complicated term, adenosine triphosphate, and even when we look at its molecular structure it seems quite involved, but if we break it down into its constituent parts it becomes a little bit more understandable and we'll begin to appreciate why, how it is a store of energy in biological systems. The first part is to break down this molecule between the part that is adenosine and the part that is the triphosphates, or the three phosphoryl groups. The adenosine is this part of the molecule, let me do it in that same color. This part right over here is adenosine, and it's an adenine connected to a ribose right over there, that's the adenosine part. And then you have three phosphoryl groups, and when they break off they can turn into a phosphate. The triphosphate part you have, triphosphate, you have one phosphoryl group, two phosphoryl groups, two phosphoryl groups and three phosphoryl groups. One way that you can conceptualize this molecule which will make it a little bit easier to understand how it's a store of energy in biological systems is to represent this whole adenosine group, let's just represent that as an A. Actually let's make that an Ad. Then let's just show it bonded to the three phosphoryl groups. I'll make those with a P and a circle around it. You can do it like that, or sometimes you'll see it actually depicted, instead of just drawing these straight horizontal lines you'll see it depicted with essentially higher energy bonds. You'll see something like that to show that these bonds have a lot of energy. But I'll just do it this way for the sake of this video. These are high energy bonds. What does that mean, what does that mean that these are high energy bonds? It means that the electrons in this bond are in a high energy state, and if somehow this bond could be broken these electrons are going to go into a more comfortable state, into a lower energy state. As they go from a higher energy state into a lower, more comfortable energy state they are going to release energy. One way to think about it is if I'm in a plane and I'm about to jump out I'm at a high energy state, I have a high potential energy. I just have to do a little thing and I'm going to fall through, I'm going to fall down, and as I fall down I can release energy. There will be friction with the air, or eventually when I hit the ground that will release energy. I can compress a spring or I can move a turbine, or who knows what I can do. But then when I'm sitting on my couch I'm in a low energy, I'm comfortable. It's not obvious how I could go to a lower energy state. I guess I could fall asleep or something like that. These metaphors break down at some point. That's one way to think about what's going on here. The electrons in this bond, if you can give them just the right circumstances they can come out of that bond and go into a lower energy state and release energy. One way to think about it, you start with ATP, adenosine triphosphate. And one possibility, you put it in the presence of water and then hydrolysis will take place, and what you're going to end up with is one of these things are going to be essentially, one of these phosphoryl groups are going to be popped off and turn into a phosphate molecule. You're going to have adenosine, since you don't have three phosphoryl groups anymore, you're only going to have two phosphoryl groups, you're going to have adenosine diphosphate, often known as ADP. Let me write this down. This is ATP, this is ATP right over here. And this right over here is ADP, di for two, two phosphoryl groups, adenosine diphosphate. Then this one got plucked off, this one gets plucked off or it pops off and it's now bonded to the oxygen and one of the hydrogens from the water molecule. Then you can have another hydrogen proton. The really important part of this I have not drawn yet, the really important part of it, as the electrons in this bond right over here go into a lower energy state they are going to release energy. So plus, plus energy. Here, this side of the reaction, energy released, energy released. And this side of the interaction you see energy, energy stored. As you study biochemistry you will see time and time again energy being used in order to go from ADP and a phosphate to ATP, so that stores the energy. You'll see that in things like photosynthesis where you use light energy to essentially, eventually get to a point where this P is put back on, using energy putting this P back on to the ADP to get ATP. Then you'll see when biological systems need to use energy that they'll use the ATP and essentially hydrolysis will take place and they'll release that energy. Sometimes that energy could be used just to generate heat, and sometimes it can be used to actually forward some other reaction or change the confirmation of a protein somehow, whatever might be the case. |
98 | Biomolecules | Overview Metabolism | ATP hydrolysis: Gibbs free energy | So let's talk about one of the most famous molecules in all of biochemistry, ATP. So why is ATP so famous? Well, it's the energy currency of the cell. And the reason it's called the energy currency of the cell is because it powers, it essentially fuels, many life sustaining reactions inside of our body. So some examples of these include biosynthesis of biomolecules. So remember fats, proteins, carbohydrates, and nucleic acids are all essential to life, and building these molecules requires energy in the form of ATP. In addition, ATP is also used to contract our muscles. And this is very important in order to allow living organisms to move. And additionally, ATP is also involved in some ion movement across cell membranes. And of course, moving ions across membranes is really important to maintain a comfortable internal environment within the cell. Now, you could take my word for it that somehow ATP magically powers all of these lifesustaining processes, but you really don't have to. In fact, in this video, we're going to review some topics from general chemistry to really understand how ATP, on a chemical level, really fuels these reactions. Now, the topic we want to review in introductory chemistry is a thermodynamic parameter called Gibbs free energy, or as it's more often written as just simply delta G. So now recall that delta G is a quantitative number. And it's a number that's measured in units of joules, which is a measurement of energy. And depending on whether this value is positive or negative, it tells us whether or not a reaction requires energy, or whether a reaction releases energy. Now remember that delta G is equal to the free energy of the products of a reaction, minus the free energy of the reactants in a reaction. So if the change in Gibbs free energy is negative, which means that the products have a much smaller free energy than the reactant, we say that this reaction releases energy. On the other hand, if we have a positive value of delta G, which means that our products are at a much higher energy level than our reactants, we say that that reaction requires an input of energy. And I know I've been kind of nebulous about this term energy here. And so, briefly, I want to remind you that the change in free energy going from reactants to products of a reaction takes into account both the change in enthalpy, as well as the change in entropy, which are two topics that you might be familiar with from general chemistry. So how does this all relate to ATP? Well, it turns out that there is a reaction involving ATP that has a very large negative delta G value. That is to say it releases a lot of free energy. Specifically, this reaction involves ATP combining with water, and when it combines with water, we call this a hydrolysis reaction. So I'll just write that here to remind us. And the products of this reaction are a molecule called ADP and a free phosphate group. And like I mentioned before, the change in Gibbs free energy is very negative. So what's going on here? So ATP starts out with triphosphate, three phosphate groups, loses a phosphate group because it becomes diphosphate. And then it forms a free phosphate group that it cleaved off. So on first glance, it might seem that this reaction is not balanced because we don't have this hydrogen or oxygen on the right side of our equation. But I just want to note here that the negatively charged hydroxyl group becomes a part of the phosphate group. And the remaining hydrogen ion of the water combines with another molecule of water in solution to become a positively charged hydronium ion. And usually, these two things are left out just for the sake of convenience. But I wanted to point them out here so that you wouldn't be confused by the stoichiometry. On the other hand, many biosynthesis reactions in the body have a positive delta G value. So remember, a positive delta G value means it requires an input of energy. And an example of this type of reaction is when we take a monomer, such as an amino acid for example, and we string them together covalently to form a polymer. So in the case of an amino acid, that would mean we're forming a long peptide chain. Now here's where our knowledge of introductory chemistry comes in. So in thermodynamics, the study of energy changes, there's an important principle that states that the overall delta G for a reaction I'm going to scroll down here to give us some more space. So the overall delta G for a reaction is equal to the sum of the delta G values for the individual steps of a reaction. So let's actually go ahead and add these two reactions together and see what happens. So let's write that out. So we have ATP as a reactant, as well as water, as well as our monomer subunits. And we are producing ADP, a free phosphate group, and a polymer. Now what is the delta G for our overall reaction? Well, we just simply have to add the delta G values for each step. Now I didn't give you actual numerical values for each of these steps. But in general, the hydrolysis of ATP produces energy in excess of the energy needed for biosynthesis reactions, such as this one. So essentially what I'm saying is that if we add a very large negative number to a smaller positive number, we will get an overall negative delta G value. In other words, we have just taken a previously energetically unfavorable reaction with a positive delta G value and turned it into an energetically favorable reaction with a negative delta G value. And we have done this by what we call coupling a reaction that has a favorable delta G value, such as the ATP hydrolysis, with a reaction that has an unfavorable delta G value. I want to mention that the ability to add these delta G values tells us nothing about the path that the reaction actually takes. And in fact, generally speaking, almost never does a reaction proceed in two discrete steps like it's written here. Instead, this coupled process often occurs simultaneously. But as you can see, it's still beneficial to separate these two reactions into two discrete steps, so you can prove to yourself essentially why ATP, with its negative delta G value, is able to fuel energetically unfavorable processes. |
99 | Biomolecules | Overview Metabolism | ATP hydrolysis: Transfer of a phosphate group | Oftentimes in biochemistry, we write energyrequiring processes such as in the conversion of a monomer to a larger polymer as onestep reactions that are ultimately fueled by a separate reaction involving the breakdown, or hydrolysis remember, hydrolysis just means reaction with water of ATP to produce ADP and a free phosphate group. Now, when we write it this way, it implies that the breakdown of ATP is entirely separate from the biosynthesis reaction that it's fueling. When in fact, in the body, coupled reactions such as these often occur simultaneously. In fact, almost always, ADP is directly involved in the chemical reaction that it's fueling by directly donating or transferring one of its phosphate groups to the reaction it's involved in. And this case, in our example, it donates a phosphate group directly to the monomer. To understand this mechanism even better, let's go ahead and take a look at a more tangible example in which we take a monomer and convert it into a polymer. So let's actually go ahead and take a look at building up nucleotides into a polynucleotide chain such as DNA. So for the sake of simplicity, I'm going to go ahead and just draw a symbol for our nucleotide instead of writing out the entire chemical formula. But let's go ahead and review what the components of a nucleotide are. So remember that a nucleotide is composed of a sugar and nitrogenous base along with a phosphate group. And I'm going to highlight this phosphate group, because it will be particularly important in the covalent bonds that form between nucleotides to form DNA. In addition, the sugars have a hydroxyl group that are also involved in these bonds. I'm going to highlight that here as well. In the very first step of the reaction, two ATP molecules make an appearance. And specifically, they donate one phosphate group each to the phosphate group already on the nucleotide. Let's go ahead and draw out what our products look like. So we have our nucleotide and our untouched hydroxyl group. But now, instead of one phosphate group, we have a total of three phosphate groups that are covalently bonded to one another. And because ATP gave up its phosphate group, we are left with two molecules of ADP. Now, there is one point of confusion when we're talking about the breakdown of ATP into ADP. Many of us fall into this habit of saying that it's the breakdown of ATP into ADP that provides the energy for a reaction. But it turns out that in this step the reaction in which we're essentially stacking up all these negatively charged phosphate groups that don't want to be next to each other has a positive delta G value and cannot drive the reaction forward. But what does drive the overall reaction forward is the second step of the reaction. So let's talk about what happens in the second step. In the second step of the reaction, in comes a growing polynucleotide chain. So I'm going to go ahead and just draw two of these nucleotides. And remember that on one end of the growing polynucleotide chain is a hydroxyl group, which I'm highlighting in green. And on the other end is a phosphate group, which I'm highlighting in blue. Bonds between the nucleotides involve both the hydroxyl group in green and the phosphate group in blue. In this step of the reaction, the hydroxyl group of the growing polynucleotide chain essentially displaces the phosphate groups that were added in the previous step of the reaction. Now, recall that this is possible because having three phosphate groups all crowded together like this makes the electrons in the bonds between the phosphate groups very energetically unstable. Think about all that negative charge that's close together that doesn't want to be. So let's go ahead and draw out what our final product looks like. And I'm going to go ahead copy and paste this polynucleotide chain so we don't have to redraw all of it. So here it is. Go ahead and erase the parts that we don't need here. And now at the hydroxyl end, which is this green end right here, we have covalently bonded our new nucleotide. And so I'm just going to color code this by noting that the nucleotide with the stripes is the one that we're adding. And this covalent bond is between the hydroxyl group as well as the phosphate group, which I'm indicating here in blue. And the other hydroxyl group remains on the other end of the nucleotide ready for another nucleotide to attach. Now, let's not forget the paired phosphate group that was displaced in forming this covalent bond. This paired phosphate group, which has a name that you don't need to remember it's called the pyrophosphate group undergoes a hydrolysis reaction, that is, reaction with water, to produce two independent phosphate groups. Now, the particular reaction mechanism isn't that relevant. But just so you know, the water essentially inserts itself asymmetrically in between these two phosphate groups and basically donates a hydroxyl group to one and a hydrogen to another. And because the split was asymmetric, these two phosphate groups still remain identical to one another. Now, the key point to takeaway here is that this reaction with water, this hydrolysis reaction, is very energetically favorable. Specifically, we say that it has a fairly large negative delta G value. And so it is this step of the reaction that ultimately drives this entire reaction forward. Now, if that doesn't fully click in your mind, let's also think about this from another perspective. Let's think about this in the perspective of equilibrium. So remember, there's a principle in chemistry called Le Chatelier's principle. And Le Chatelier's principle says that a system, such as a chemical reaction, will respond to a change in the system, such as a change in temperature or change in concentrations of the reactants or products, by shifting in the direction that counters that change. So in this case, what is the change in our system? Well, the change our system is that we are degrading our products. So remember, this phosphate group, this pyrophosphate group, is being broken down into individual phosphate groups. And so essentially what we're doing is we are removing the products of our reaction. And if we do this, the reaction will shift in the forward direction to produce more products. And the shift in the forward direction pushes this reaction forward. So ultimately, the reason I wanted to go through this mechanism with you was to really show you that ATP provides energy to a reaction by donating one of its phosphate groups or transferring one of its phosphate groups, and not through a simple hydrolysis reaction, even though that's often how it's written for convenience. Now, one question that I had about ATP hydrolysis when I was learning it, was that if this overall reaction here, this coupled reaction, is energetically favorable, so this overall reaction has a negative delta G value, why doesn't ATP just donate its phosphate groups to any substrate in the body? Because ATP is floating around in an aqueous solution, so potentially it could donate its phosphate group to water and expend a lot of wasteful energy. The answer to this question involves reminding ourselves of the two factors that control whether or not a reaction will occur. So the first one is energetic. So is this reaction energetically favorable? And we usually look at the value of delta G. In this case, our reaction is energetically favorable because delta G is less than 0. But the other factor that controls whether or not a reaction will occur is kinetics. And we usually talk about a parameter called the activation energy, which I'm going to abbreviate here as E sub A. And it turns out, in this example, ATP has a very high activation energy. And I'm just going to skim the surface of the topic since our video's devoted just to this topic alone. But essentially, we say that this reaction is kinetically stable because it has a very high activation energy. And the way that our body deals with this is that our body has enzymes that can lower the activation energy and allow the reaction to proceed when it should precede. And so in our example, each step of the reaction has a specific enzyme that facilitates or catalyzes each step of the reaction. And this is actually very beneficial for the body, because by having an energetically favorable but kinetically stable reaction, the body can selectively control whether or not energy will be used by controlling the enzymes that are available for the reaction to proceed. And so it's a very elegant system. And I hope that ultimately, this entire video has given you a better picture of how ATP fuels energy requiring processes on a molecular level. |
100 | Biomolecules | Overview Metabolism | Oxidation and reduction review from biological point-of-view | What I want to do in this video is review what we learned from our chemistry classes about oxidation and the opposite of oxidation, reduction. And then see how what we learned in our chemistry class relates to the way that a biologist or biochemist might use these words. And hopefully we'll see that they're the same thing. So just as a bit of review, if you watched the chemistry playlist. Oxidation, you can view it and actually there's a famous mnemonic for it. It's: OIL RIG Where the oil tells us that oxidation is losing I put it in quotes because you're not necessarily losing the electrons; I'll show you what I mean is losing electrons. This is what you should have learned in your chemistry class. And then you also learned that reduction is gaining. And I'll put that in quotes as well. Is gaining electrons. And I put that in quotes because you're not necessarily gaining electrons. You're more hogging it. And the reason why it's called reduction, is because if you are gaining electrons your notional charge, if you really were gaining them, is being reduced. And the reason why this is called oxidizing is because you tend to lose electrons to oxygen. Although it doesn't have to be oxygen. It could be any molecule that will hog electrons away from you. And I think a nice example would be fair to kind of make this a little bit more concrete. Let's say I took some molecular hydrogen, it's in a gaseous state, and I were to combust that with some molecular oxygen. This is what happened on the Hindenburg. They filled a balloon full of hydrogen and you get a little bit of spark, expose it to oxygen, and you're going to have a big explosion. But in the process, for every mole of molecular oxygen, if you have two moles of molecular hydrogen I'm just making sure the equation is balanced you're going to produce two moles of H2O plus a ton of heat. This thing is really going to blow. What I want to do, I mean we could talk about the Hindenburg but really, the whole reason why I even wrote this is, I want to show you what is getting oxidized and what is getting reduced. So in this situation right here on the hydrogen, the molecular hydrogen just looks like this. You have a hydrogenhydrogen bond. They're each sharing an electron with the other one so that they both can pretend their 1s orbital is completely filled. So they're not losing electrons to each other. They're not hogging electrons one from the other. So we say that they have a neutral oxidative state. They haven't gained or lost electrons. They're just sharing them. And the same thing is true for the molecular oxygen. And here you actually have a double bond with the two oxygens. But they're both oxygens, so there's no reason why one would gain or lose electrons from the other. But when you go on this side of the equation, something interesting happens. You have, for every oxygen is connected to two hydrogens. And the way to think about is that oxygen is hogging each of these hydrogen's electrons. So hydrogen has this one electron on its valence shell. The deal with most covalent bonding is, hey, I give you an electron, you give me an electron and we both have a complete pair. But we know, or hopefully we can review, that oxygen is much more electronegative than hydrogen. This is a little bit of glucose that's left over from our cellular restoration video. You can ignore it for now but I'm going to connect all this in a future video. But if we look at our periodic table, if you remember from the chemistry playlist, electronegativity increases as we go to the top right of the periodic table. These are the most electronegative elements over here, these are the least electronegative. And all electronegative means is, likes to hog electrons. So even though oxygen and hydrogen are in a covalent bond in water they're sharing electrons oxygen is more electronegative, much more electronegative than hydrogen, so it's going to hog the electrons. And actually if you take some elements on this side and you bond them with some guys over here, these guys are so much more electronegative than these lefthand elements that they'll actually completely steal the electron, not just hog it for most of the time. But when you talk electronegativity, it just means, likes the electrons. So when you look at this bond between hydrogen and oxygen, we saw from the periodic table, oxygen is a lot more electronegative, so the electrons spend a lot more time on oxygen. We learned about hydrogen bonding. We learned that it creates a partial negative charge on that side of the water molecule and creates partial positive charges on this side. And electrons still show up around the hydrogens every now and then. When you talk about oxidation and reduction you say, look there's no partial charge. If one guy is kind of hogging the electron more, for the sake of oxidation states, we're going to assume that he took the electron. So for an oxidation state, we'll assume that the oxygen in water takes the electron and we'll give him an oxidation state of one minus. Or the convention is, you write the charge after the number for oxidation states. So you don't confuse it with actual charges. So this has a one minus because, from an oxidation state point of view, it's taking the electron. It's gaining the elctron. That's why I put it in quotes. Because you're not really gaining it. You're just gaining it most of the time. You're hogging electrons. And likewise, this hydrogen let me be careful, this isn't he got one electron from this hydrogen and you got another electron from this hydrogen. So instead of saying one minus, it should be two minus. It should be two minus, because he's hogging one electron from here and one electron from there. And in general, when oxygen is bonding with other nonoxygen atoms or nonoxygen elements, it tends to have a two minus or a negative two oxidation state. So if this guy's two minus, because he's gained two electrons. Let me write that in quotes. Gained two electrons. We know that he really didn't gain them, that he's just hogging them. These guys lost an electron each. So this guy's oxidation state is going to be one plus. And this guy's oxidation state is going to be one plus. So you could say, by combusting the hydrogen with the oxygen, that the hydrogens before they had a zero oxygen state, each of these hydrogens had a zero oxygen state now they have a one plus oxidation state because they lost their electrons when they bonded with the oxygen. So we say that these hydrogens have been oxidized. So, due to this reaction, hydrogen has been oxidized. Why has it been oxidized? Because before, it was able to share its electrons very nicely. But then it bonds with oxygen, which will hog its electrons. So the hydrogen is losing its electrons to the oxygen, so it's been oxidized. Similarly, the oxygen, due to this combustion reaction, has been reduced. Why has it been reduced? Here it was just sharing electrons. It wasn't losing or gaining it. But here when it's bonded with an element with much lower electronegativity, all of a sudden it can start hogging the electrons, it gains electrons. So this hypothetical charge is reduced by two. And if I wanted to actually account for all of the electrons, because we're talking about losing electrons and gaining electrons, we can write two half reactions. This should all be a little bit of review from your chemistry class. But it never hurts to see this again. I'm going to throw this in the biology playlist so that you biology people can hopefully refresh your memory with this stuff. We can write two half reactions. We could say that we started off with two moles of molecular hydrogen. And they have no oxidation states, or they're neutral. So I could write a zero there if I want. And then I end up with on the other side I end up with two moles of H2. But each of the hydrogens now, have a plus one oxidation state. Or another way to think about it is, each of these there's four hydrogens here. This is molecular hydrogen has two hydrogens and we have two moles of this. So there are four hydrogens here. Each of the four hydrogens lost an electron. So I can write this. So, plus four electrons. That's the half reaction for hydrogen. It lost four electrons. So this is another way of saying that hydrogen is oxidized because it lost electrons. OIL: oxidation is losing. And then the other half reaction, if I were to write the oxygen. So I'm starting with a mole of molecular oxygen and I'm adding to that four electrons. I can't make electrons out of nowhere. I'm getting the electrons from the hydrogen, I'm adding to the oxygen. And so the half reaction on this side, I end up with two moles I could write it like this two moles of oxygen. And each of them have an oxidation state of two minus. So these are the half reactions. And all this is showing is that the hydrogen, over the course of this combustion reaction, lost electrons. And that the oxygen gained the electrons that the hydrogen lost. So this tells us that oxygen is reduced. Now this is all fair and good and this is all a bit of review of what you learned in chemistry class. But now I'm going to make things even more confusing. Because I'm going to introduce you to how a biologist thinks about it. So and it's not always the case. Sometimes the biologist will use the definition you learned in your chemistry class. But a biologist or many times in many biology textbooks they'll say and this used to confuse me to no end, really that oxidation is losing hydrogen atoms. And reduction is gaining hydrogen atoms. And at first when I got exposed to this, I was like, I learned it in chemistry class and they talk about electrons. Hydrogen atoms, you know it's a proton and an electron, how does it relate? And the reason why these two definitions this is really the whole point of this video the reason why this definition is consistent with this one is because in the biological world hydrogen is what tends to get swapped around. And it tends to bond with carbon, oxygen, phosphorous, nitrogen. And if we look at the periodic table, and we see where hydrogen is, and we see where carbon, nitrogen, oxygen and phosphorous and really all this other stuff is, you see that all of the stuff that in biological systems, hydrogen tends to bond with, the things it tends to bond with are much, much more electronegative. So if a carbon is bonding with a hydrogen, the carbon is hogging that electron. And then if that hydrogen gets transferred to an oxygen, along with the electron, the carbon will lose the hydrogen atom, but it really lost the electron that it was hogging before. And now the oxygen can hog that electron. So these are really consistent definitions. And the whole reason why I showed you this example is because the biological definition doesn't apply here. I mean, you could say, well, oxygen is definitely gaining hydrogens in this reaction. So we can definitely say that oxygen is being reduced still, according to the biological definition. But you can't really say that hydrogen is losing hydrogens here. In this situation, hydrogen is just losing electrons. It's not losing itself. I guess you could say it's losing itself because it's being taken over. But the biological definition just comes from the same notion. That when hydrogen bonds with most things in biological compounds, it tends to give the electrons. So if a carbon loses a hydrogen and gives it to an oxygen, the carbon will lose that hydrogen's electron that it was able to hog. And now the oxygen is hogging it. So the carbon would be oxidized and the oxygen would be reduced. Hope that doesn't confuse you. In the next video I'll show you a couple more examples. And the whole reason why I'm doing this is to apply this to cellular respiration. So that you don't get confused when people talk and say that, oh the NAD is being reduced when it picks up the hydrogen. Or it's being oxidized when it loses the hydrogen, and so forth and so on. I wanted you to see that these are the same definitions that you learned in your chemistry class. |