Lecture 13: Gene Expression Pt. 1

Today:

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• Intro/review of gene expression
• Intro/review of gene regulation in bacteria
• Setting up what we will need for a mathematical model describing the dynamics of gene expression
• E.g. how does the concentration of proteins for a given gene change if an inhibitor starts being produced
• Next time we’ll go through how we’ll actually write out the math

Miller, et al. (1970)

Neurons (credit Wikipedia)

Short digression to humans (!) . . .

Cardiac cells (Vunjak-Novaković Lab)

Bone cells

de Peppo, et al. (2013)

Cancer cells

Emert, et al. (2021)

Et c . . .

Bacillus subtilis cells

Süel, et al. (2006)

spore

competent cell

motility

matrix

membrane potential

How are these genetically identical cells all so different?

They’re making a different subset of their available genes!

B. Subtilis cell differentiation network

Lopez, et al. (2008)

competent cell

spore

motile cell

matrix producer

The dynamics of all these genes tell you

• When you will become each cell type
• How long you’ll stay that type
• Etc, etc

E. coli switching metabolism

E. coli

eating glucose

eating xylose

How does a single-celled organism switch from one metabolic pathway to another?

All gene expression and gene regulation!

The genome contains a list of genes; how they interact determines how the cell works.

Monod (1966)

Our starting point: the central dogma of biology

DNA

gene

DNA

RNA

protein

transcription

translation

Operons

In prokaryotes, often multiple proteins with related functions are transcribed as a single continuous RNA. Such a structure is called an operon.

Shafee and Lowe (2017)

The Central Dogma of Biology

DNA

mRNA

protein

transcription

translation

enzymes

transporters

RNA polymerase

DNA polymerase

ribosomal proteins

structural proteins

transcription factors

tRNA

rRNA

CRISPR RNA

C, N, PO₄, …

ribosomes

amino acids, NTPs, dNTPs, …

replication

kinases

Et cetera . . .

How do we make sense of this?

Herrgård, et al. (2003)

Known E. coli gene regulatory network as of 2003

How do we make sense of this?

Herrgård, et al. (2003)

What we’ll do over the next few weeks:

• Identify the most common regulatory network structures here
• Figure out how to measure the different parts of the network
• Build simple models for them based on the central dogma
• See how this complicated system is made from several very common repeated motifs

First, how are bacterial genes regulated?

promoter

protein coding sequence

gene

RNA polym.

repressor

activator

protein

The most common mechanisms:

mRNAs can be destroyed

proteins can be destroyed

Transcription can be repressed by a protein

Transcription can be activated by a protein

Seems mostly much simpler than in eukaryotes, where there are many, many regulatory DNA sequences, post-transcriptional modifications, etc.

mRNA

Our basic framework

DNA

mRNA

protein

For a given gene:

Let [m] by the concentration of the mRNA for the gene and [P] be the concentration of the protein.

How to we describe the dynamics of these molecules within the cell?

transcription of DNA into RNA

active degradation of mRNA, dilution due to growth

translation of mRNA into protein

active degradation of protein, dilution due to growth

Can be influenced by the level of another gene or itself through gene regulation!

[m]

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[P]

What do we need to build and test a model?

We need to know several rates to set parameter values in our equations

• What are the rates of mRNA production from DNA?
• What are the rates of mRNA loss?
• What are the rates of protein production from mRNAs?
• What are the rates of protein loss?

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Then we need to know

• How do we incorporate those rates into the equations?
• How do we represent gene regulation mathematically?

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How do we measure gene expression in cells to compare to models?

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Today!

After spring break!

What are the rates of mRNA transcription?

An experiment to measure this rate:

Gotta, et al. (1991)

1000 bp

Electron microscope image of DNA from extracted from live E. coli

rRNA operon

direction of transcription

nascent RNA transcripts

DNA

What are the rates of mRNA transcription?

An experiment to measure this rate:

Gotta, et al. (1991)

1000 bp

DNA from E coli after 40 seconds of rifampin exposure, a drug that inhibits transcription initiation, but not RNA elongation

continuing transcription

untranscribed DNA

Treat E. coli w/transcription initiation inhibitor; extract DNA

What are the rates of mRNA transcription?

An experiment to measure this rate:

Gotta, et al. (1991)

1000 bp

After 70 seconds

continuing transcription

untranscribed DNA

What are the rates of mRNA transcription?

An experiment to measure this rate:

Gotta, et al. (1991)

3. Measure how far on average the last RNA polymerase has moved

What are the rates of mRNA transcription?

An experiment to measure this rate:

Gotta, et al. (1991)

On average, 42 nucleotides per second transcribed.

Collecting other experimental results, the rate of transcription in bacteria is

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40-80 nt/s

How long does it take to transcribe a gene?

DNA

mRNA

~1000 nt

1000 nt, ~60 nt/s:

10-20 seconds to transcribe a gene

RNA polymerase

How long?

What are the rates of protein synthesis?

An experiment to measure this rate:

Young and Bremer (1976)

1. Grow E. coli with radioactive ¹⁴C-leucine
2. Add small amount of radioactive ³H-leucine
3. At regular time points, extract protein from cells, isolate specific proteins with gel electrophoresis, and measure ³H/¹⁴C ratio
4. As more protein gets synthesized, a higher proportion of the protein will have the radioactive hydrogen label.
5. Make the measurement for proteins of many different molecular weights to estimate synthesis rate

What are the rates of protein synthesis?

An experiment to measure this rate:

Young and Bremer (1976)

³H / ¹⁴C ratio

Proteins w/3 different molecular weights

What are the rates of protein synthesis?

An experiment to measure this rate:

Young and Bremer (1976)

glucose + amino acids

glucose only

succinate

17 amino acids/sec for fast growth

12 amino acids/sec for fast growth

Let’s compare mRNA and protein synthesis rates

mRNA: ~60 nt/sec

protein: ~17 a.a./sec

The rates are roughly matched!

Let’s compare mRNA and protein synthesis rates

Miller, et al. (1970)

Actively transcribed mRNAs are actively translated!

Now for mRNA and protein “loss”.

How is mRNA “lost”? What are the rates of loss?

Ribonuclease (RNase) enzymes actively degrade RNAs.

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How quickly does this happen to an mRNA in a cell?

What are the rates of degradation?

Measuring mRNA decay in E. coli:

Bernstein, et al. (2002)

Grow E. coli

Extract RNA

Convert to DNA with fluorescent labels

Measure mRNA levels with a gene microarray chip

What are the rates of degradation?

To measure decay rate/half life:

Bernstein, et al. (2002)

2. Measure mRNA levels

1. Add transcription inhibitor rifampicin for a controlled period of time

3. Repeat for increasing periods of rifampicin exposure. During this time there is no new transcription, only degradation

4. Measure mRNA half lives

What are the half lives of mRNAs?

Bernstein, et al. (2002)

Very short! Most genes degraded to half their concentration in less than 6 minutes!

Interesting note from these authors: no correlation observed between mRNA half life and 1) abundance, 2) secondary structure, or 3) cell growth rate.

Note: these half lives reflect much faster degradation than would be accounted for by dilution due to cell growth.

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Active degradation is the dominant factor for mRNA loss!

How is protein “lost”? What are the rates of loss?

An experiment to measure protein decay in E. coli:

growing bacteria

Filter that allows media through, but not cells

Add non-radio leucine

Nath and Koch (1970)

1. Grow bacteria with radioactively labeled leucine
• Leucine easily taken up by cells and not broken down into constituents
2. Put bacteria in flow cell
3. Add non-radioactive leucine
4. Let non-radioactive leucine displace free radioactive leucine
5. At time intervals, measure radioactivity of the media coming through the filter (“perfusate”).
6. If cells are not dying or growing quickly, the protein in the perfusate will reflect levels of degraded protein

Measure radioactivity of run-off normalized to initial cell radioactivity

All performed under slow metabolic conditions with little cell growth

flow cell

How is protein “lost”? What are the rates of loss?

Nath and Koch (1970)

Measured rate of radioactivity of perfusate

Fast component of protein degradation

Slow component of protein degradation

How is protein “lost”? What are the rates of loss?

Nath and Koch (1970)

They are able to estimate two things:

1. The half-life of the fast component
2. The percentage of total protein the

Findings:

• Average half-life for fast component is ~60 minutes
• Fast component accounts for 5-7% of total protein
• Under some conditions as low as 2%!

Conclusion: active, rapid degradation is not a major component of protein loss in E. coli.

To summarize

DNA

mRNA

protein

Similar synthesis rates

Loss is fast; mainly due to active degradation by RNases

Loss is slow; mainly due to dilution from growth

What will these measurements tell us?

DNA

mRNA

protein

active degradation

transcription

translation

dilution due to cell growth

active degradation

dilution due to cell growth

Thinking about concentrations in the cell:

similar rates

dominant factor

dominant factor

What will these measurements tell us?

DNA

mRNA

protein

transcription

translation

Depending on regulation, this will predict:

• How protein level responds to changes in mRNA level
• The dynamics of gene regulation
• A gene the regulates itself
• Genes that regulate other genes

In order to test these predictions, we have to measure gene expression. How do we do that?

How do you measure gene expression?

1. Western blot, protein-level

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Pain in the ass, but still the gold standard

(Wikipedia)

How do you measure gene expression?

2. qPCR, RNA-level

RNA

Complementary DNA

Kill and permeabilize cells

Extract DNA and perform pcr on gene of interest

How do you measure gene expression?

3. RNA sequencing, RNA-level

RNA

Complementary DNA

Kill and permeabilize cells

Extract DNA and sequence

How do you measure gene expression?

4. Mass spec proteomics, protein-level

Mass spectrometry

1. Take a sample of cells and extract proteins�����
2. Ionize proteins�����
3. Run ionized proteins through an electric field to determine masses
4. Compare masses to known masses of proteins

How do you measure gene expression?

5. RNA fluorescence in situ hybridization (FISH), RNA-level

RNA

Complementary DNA probe with fluorescent label

Cell/sample

How do you measure gene expression?

4. RNA fluorescence in situ hybridization (FISH), RNA-level

mRNA 1

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mRNA 2

(Wikipedia)

How do you measure gene expression?

6. Fluorescent proteins

First, a quick digression on fluorescence!

motility

matrix

Fluorescence

absorb photon/light

electronic state/”orbital”

Fluorescein (FITC)

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(fluorescent molecule, “fluorophore”)

• Electron absorbs light energy, going to higher, unstable state
• Electron loses energy through heat/”vibration” without emitting light
• Electron loses the rest of its energy by emitting light → fluorescence!
• Because the radiative energy loss is less than the original gain, the wavelength/color is different! This is called a Stokes shift

Higher energy photon have shorter wavelength (more ~blue); lower energy photons have longer wavelength (more ~red)

Fluorescent proteins

Measuring bacterial gene expression with fluorescent proteins

gene of interest: tapA

tasA

protein coding sequence

P_tapA

6. Method 1: transcriptional reporters

promoter

Same regulatory sequence. gfp is regulated the same way as tasA in this cell. If you seen no GFP signal in a cell, it’s probably not making a lot of tasA. If you see a lot of signal, it’s probably making a lot.

Measuring bacterial gene expression with fluorescent proteins

1. Method 1: transcriptional reporters

P_citZ-YFP

(B. subtilis)

Measuring bacterial gene expression with fluorescent proteins

gene of interest: tasA

6. Method 2: fluorescent protein fusion

Create a mutant with:

The protein is made with a fluorescent protein physically connected.

Only used when spatial protein localization is strictly needed because attaching GFP changes a protein’s behavior in unknown ways!

Ko and Lee (2019)