Regulation of Gene Expression

LAC OPERON

TRYPTOPHAN OPERON

Gene regulation

• For a cell to function properly, necessary proteins must be synthesized at the proper time.
• All cells control or regulate the synthesis of proteins from information encoded in their DNA.
• The process of turning on a gene to produce RNA and protein is called gene expression.
• Whether in a simple unicellular organism or a complex multi-cellular organism, each cell controls when and how its genes are expressed.
• For this to occur, there must be a mechanism to control when a gene is expressed to make RNA and protein, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed.

A schematic showing a protein coding gene and some of the questions or problems that we need to ask ourselves or alternatively problems we need to know solutions for if we are to understand how regulation of the transcriptional portion of the gene's expression is regulated.

Gene regulation – two types

Positive regulation:

• The gene regulation is said to be positive when its expression is increased by a regulatory element (positive regulator)

Negative regulation:

• A decrease in the gene expression due to the presence of a regulatory element (negative regulator) is referred to as negative regulation.

Constitutive and inducible genes

Constitutive genes (or housekeeping genes) :

• The products (proteins) of these genes are required all the time in a cell. Therefore, the constitutive genes (or housekeeping genes) are expressed at more or less constant rate in almost all the cells and, further they are not subjected to regulation.
• Eg. The enzymes of citric acid cycle.

Inducible genes:

• The concentration of the proteins synthesized by inducible genes is regulated by various molecular signals. An inducer increases the expression of these genes while a repressor decreases.
• Eg. Tryhtophan pyrrolase of liver is induced by tryptophan.

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One cistron-one subunit concept

• Cistron – is the smallest unit of genetic expression.
• It is the fragment of DNA coding for the subunit of a protein molecule.
• The original concept of one gene-one enzyme is replaced by one cistron-one subunit.

Models for the study of gene expression

THE OPERON CONCEPT

Operon ?

• Operon is a cluster of genes which have a common promoter, they are transcribed as a single mRNA.
• Examples of operon include,
• lac operon
• trp operon

It was Jacob and Monod in 1961 who proposed the operon model for the regulation of transcription.

Lac Operon

• lac operon is an inducible operon present in Escherichia coli.
• It encodes the genes involved in the catabolism of lactose.
• It is induced by the sugar lactose or allolactose.
• It has three structural genes like lac z, lac y and lac a and three regulatory genes like promoter, operator and repressor.

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Lac Operon

• ● lac z : It codes for β - galactosidase enzyme and hydrolyzes lactose into glucose and galactose.

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• ● lac y: It codes for permease enzyme and it increases the permeability of the cell to lactose.

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• ● lac a: It codes for transacetylase enzymes and it transfers acetyl group to Β - galactosidase.

Lac Operon

• Operator gene:
• It is present adjacent to the structural genes and directly controls the synthesis of mRNA, over the structural genes. It interacts with the repressor protein and prevents the transcription of structural genes.

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• Promoter gene:
• Promoter gene is the site for initial binding of RNA polymerase.

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• Regulator gene:
• Regulator gene codes for repressor protein. It produces a repressor that binds to the operator gene and stops the working of the operator.

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Lac Operon

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• Repressor:
• Repressor is a protein produced by the regulator gene and it binds to the operator gene, so that the transcription of mRNA structural genes stops.

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• Inducer:
• Inducer is a chemical like hormone or metabolite which after coming in contact with the repressor forms an inducer repressor complex. This complex cannot bind with the operator gene, which is thus switched on.

Structure of Lac operon

Lac Operon

• In the lac operon, the structural genes are the lacZ, lacY and lacA genes encoding _-galactosidase, the permease, and the transacetylase, respectively.
• Transcription occurs from a single promoter (P_lac) that lies upstream of these structural genes and binds RNA polymerase.
• However, also present are an operator site (O_lac) between the promoter and the structural genes, and a lacI gene that codes for the lac repressor protein.
• The lacI gene has its own promoter (PlacI) that binds RNA polymerase and leads to transcription of lac repressor mRNA and hence the production of lac repressor protein monomers.
• Four identical repressor monomers come together to form the active tetramer which can bind tightly to the lac operator site, O_lac.

Inducers and the Induction of Lac operon

• Normally, E. coli cells make very little of any of these three proteins but when lactose is available it, causes a large and coordinated increase in the amount of each enzyme.
• Thus each enzyme is an inducible enzyme and the process is called induction.
• The mechanism is that the few molecules of ß-galactosidase in the cell before induction convert the lactose to allolactose which then turns on the transcription of these three genes in the lac operon.
• Thus allolactose is an inducer.
• Another inducer of the lac operon is isopropylthiogalactoside (IPTG). Unlike allolactose, this inducer is not metabolized by E. coli and so, is useful for experimental studies of induction only.

Switch on mechanism of lac operon:

• When lactose is present in the growth medium, it enters into the cell by the action of permease.
• The low level of expression of lac operon is necessary for admitting the lactose into the cells.
• The lactose acts as an inducer and binds to repressor protein.
• It inactivates the repressor and allows the RNA polymerase to bind on the promoter region.
• Now the lac operon is switched on.
• This is positive regulation.

Switch off mechanism of lac operon:

• Normally, the repressor protein binds to the operator region, blocking the RNA polymerase and preventing the transcription.
• Now the lac operon is switched off.
• This is negative regulation.

Lac Operon in absence of Inducers

• In the absence of an inducer such as allolactose or IPTG, the lacI gene is transcribed and the resulting repressor protein binds to the operator site of the lac operon, Olac, and prevents transcription of the lacZ, lacY and lacA genes.

Lac Operon in presence of Inducers

• During induction, the inducer binds to the repressor.
• This causes a change in the conformation of the repressor that greatly reduces its affinity for the lac operator site.
• The lac repressor now dissociates from the operator site and allows the RNA polymerase (already in place on the adjacent promoter site) to begin transcribing the lacZ, lacY and lacA genes.
• They are transcribed to yield a single polycistronic mRNA that is then translated to produce all three enzymes in large amounts.
• The existence of a polycistronic mRNA ensures that the amounts of all three gene products are regulated coordinately.
• If the inducer is removed, the lac repressor rapidly binds to the lac operator site and transcription is inhibited almost immediately.

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CRP/CAP

• High-level transcription of the lac operon requires the presence of a specific activator protein called catabolite activator protein (CAP), also called cAMP receptor protein (CRP).
• This protein, which is a dimer, cannot bind to DNA unless it is complexed with 3’5′ cyclic AMP (cAMP).
• The CRP–cAMP complex binds to the lac promoter just upstream from the binding site for RNA polymerase.
• It increases the binding of RNA polymerase and so stimulates transcription of the lac operon.
• Whether or not the CRP protein is able to bind to the lac promoter depends on the carbon source available to the bacterium.

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Lac operon in the presence of Glucose

• When glucose is present, E. coli does not need to use lactose as a carbon source and so the lac operon does not need to be active.
• Thus the system has evolved to be responsive to glucose.
• Glucose inhibits adenylate cyclase, the enzyme that synthesizes cAMP from ATP.
• Thus, in the presence of glucose the intracellular level of cAMP falls, so CRP cannot bind to the lac promoter, and the lac operon is only weakly active (even in the presence of lactose).

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Lac operon in the absence of Glucose

• When glucose is absent, adenylate cyclase is not inhibited, the level of intracellular cAMP rises and binds to CRP.
• Therefore, when glucose is absent but lactose is present, the CRP–cAMP complex stimulates transcription of the lac operon and allows the lactose to be used as an alternative carbon source.
• In the absence of lactose, the lac repressor, of course, ensures that the lac operon remains inactive.
• These combined controls ensure that the lacZ, lacY and lacA genes are transcribed strongly only if glucose is absent and lactose is present.

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Positive and Negative Regulation of Lac Operon

• The lac operon is a good example of the negative control (negative regulation) of gene expression in that bound repressor prevents transcription of the structural genes.
• Positive control or regulation of gene expression is when the regulatory protein binds to DNA and increases the rate of transcription.
• In this case, the regulatory protein is called an activator. The CAP/CRP involved in regulating the lac operon is a good example of an activator.
• Thus the lac operon is subject to both negative and positive control.

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Tryptophan (Trp) Operon �-A Repressor Operon

Tryptophan (Trp) Operon

• Many protein-coding genes in bacteria are clustered together in operons which serve as transcriptional units that are coordinately regulated.
• It was Jacob and Monod in 1961 who proposed the operon model for the regulation of transcription.
• The operon model proposes three elements:
• A set of structural genes (i.e. genes encoding the proteins to be regulated);
• An operator site, which is a DNA sequence that regulates transcription of the structural genes.
• A regulator gene which encodes a protein that recognizes the operator sequence.
• Operons are thus clusters of structural genes under the control of a single operator site and regulator gene which ensures that expression of the structural genes is coordinately controlled.

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Trp Operon

• The tryptophan operon is the regulation of transcription of the gene responsible for biosynthesis of tryptophan.
• The tryptophan (trp) operon contains five structural genes encoding enzymes for tryptophan biosynthesis with an upstream trp promoter (Ptrp) and trp operator sequence (Otrp).
• Structural genes are TrpE, TrpD, TrpC, TrpB and TrpA
• trpE: It enodes the enzyme Anthranilate synthase I
• trpD: It encodes the enzyme Anthranilate synthase II
• trpC: It encodes the enzyme N-5’-Phosphoribosyl anthranilate isomerase and Indole-3-glycerolphosphate synthase
• trpB: It encodes the enzyme tryptophan synthase-B sub unit
• trpA: It encode the enzyme tryptophan synthase-A sub unit
• The trp operator region partly overlaps the trp promoter.
• The operon is regulated such that transcription occurs when tryptophan in the cell is in short supply.

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In the Absence of Tryptophan

• In the absence of tryptophan, a trp repressor protein encoded by a separate operon, trpR, is synthesized and forms a dimer.
• However, this is inactive and so is unable to bind to the trp operator and the structural genes of the trp operon are transcribed.

In the Presence of Tryptophan

• When tryptophan is present, the enzymes for tryptophan biosynthesis are not needed and so expression of these genes is turned off.
• This is achieved by tryptophan binding to the repressor to activate it so that it now binds to the operator and stops transcription of the structural genes.
• Binding of repressor protein to operator overlaps the promoter, so RNA polymerase cannot bind to the prometer. Hence transcription is halted.
• In this role, tryptophan is said to be a co-repressor. This is negative control, because the bound repressor prevents transcription.

Trp Operon Attenuation

Trp Operon Attenuation

• A second mechanism, called attenuation, is also used to control expression of the trp operon.
• The 5′ end of the polycistronic mRNA transcribed from the trp operon has a leader sequence upstream of the coding region of the trpE structural gene.
• This leader sequence encodes a 14 amino acid leader peptide containing two tryptophan residues.
• The function of the leader sequence is to fine tune expression of the trp operon based on the availability of tryptophan inside the cell.

Trp Operon Attenuation

• The leader sequence contains four regions (numbered 1–4) that can form a variety of base paired stem-loop (‘hairpin’) secondary structures.
• The regions are: Region 1, region 2, region 3 and Region 4. Region 3 is complementary to both region 2 and region 4.
• If region 3 and region 4 base pair with each other, they form a loop like structure called attenuator and it function as transcriptional termination. If pairing occur between region 3 and region 2, then no such attenuator form so that transcription continues.
• Attenuation depends on the fact that, in bacteria, ribosomes attach to mRNA as it is being synthesized and so translation starts even before transcription of the whole mRNA is complete.

When Trytophan is abundant

When Trytophan is abundant

• When tryptophan is abundant, ribosomes bind to the trp polycistronic mRNA that is being transcribed and begin to translate the leader sequence.
• Now, the two trp codons for the leader peptide lie within sequence 1, and the translational Stop codon lies between sequence 1 and 2.
• During translation, the ribosomes follow very closely behind the RNA polymerase and synthesize the leader peptide, with translation stopping eventually between sequences 1 and 2.
• At this point, the position of the ribosome prevents sequence 2 from interacting with sequence 3.
• Instead sequence 3 base pairs with sequence 4 to form a 3:4 stem loop which acts as a transcription terminator.
• Therefore, when tryptophan is present, further transcription of the trp operon is prevented.

When Trytophan is scarce

When Trytophan is scarce

• If, however, tryptophan is in short supply, the ribosome will pause at the two trp codons contained within sequence 1.
• This leaves sequence 2 free to base pair with sequence 3 to form a 2:3 structure (also called the anti-terminator), so the 3:4 structure cannot form and transcription continues to the end of the trp operon.
• Hence the availability of tryptophan controls whether transcription of this operon will stop early (attenuation) or continue to synthesize a complete polycistronic mRNA.

Regulation of Trp Operon

• Overall, for the trp operon, repression via the trp repressor determines whether transcription will occur or not and attenuation then fine tunes transcription.