Business of the day

Today: Ch. 18 Regulation of Gene Expression

Chapter 18 homework due next Thursday at 9 am

Homework Ch 13 and 14 due today at 9 am, Ch 15 due Tuesday at 9 am.

Exam: one week from today here at class time; Ch. 12-18

Next help session this afternoon at 5 pm in Blake

Special help session on Sunday at 5 pm in 214 Foster—come with questions

Chapter 18: Regulation of Gene Expression

Genes and metabolic pathways need to be turned on and off at appropriate times. Otherwise, organisms would be making unnecessary products and wasting resources.

Regulation of gene expression refers specifically to the cellular mechanisms that turn genes on and off or control the appearance and longevity of gene products (like proteins).

Metabolic control: This is somewhat different. It involves turning gene products (mostly enzymes) on and off.

E. coli in the gut have no control over the host’s diet (and they eat what the host eats), so they may have to manufacture certain nutrients themselves (e.g., the amino acid tryptophan--Trp)

Regulation of Bacterial Gene Expression

There are two ways to control the production of Trp

1. Metabolic control: Control the metabolic pathway that makes Trp (control enzymes)

2. Control of gene expression: Control transcription of enzymes that help build Trp

The Tryptophan anabolic pathway requires several steps, each mediated by an enzyme

Trp can control its own production by feedback inhibition

If there is lots of Trp available to a bacterium, it will inhibit the anabolic pathway that makes Trp by interfering with enzyme 1.

1. Metabolic control turns enzymes (and thus enzyme pathways) on or off quickly

Metabolic control is based on feedback inhibition(negative feedback)

An enzyme’sallostericsite

Positive Feedback: sometimes binding to an allosteric site increases enzyme activity and increases the rate of production (we will see this with the Lac Operon later in this chapter).

From Ch. 6: The end product inhibits an enzymatic pathway

2. In addition to metabolic control, regulation of gene expression can be used to control the production of Trp

Bacteria can turn on (or shut down) the genes that produce enzymes. This is almost always done by controlling transcription.

Gene Regulation: Controlling transcriptionIn bacteria, genes are often located in operons (Jacob & Monod 1961)Operons consist of several genes controlled by one promoter. Inhibiting (or stimulating) that promoter slows (or speeds up) mRNA

production

start and stop codons cause each gene to be translated separately into polypeptides

The operator determines whether RNA polymerase can proceed

Trp Operon Negative Control: Trp shuts down trp operon

Tryptophan is an amino acid produced with the help of a series of enzymes. These enzymes are encoded by genes in the Tryptophan Operon. Transcription of the genes in the Trp operon is turned off by a protein called a repressor, which sits on the Trp operator and blocks RNA polymerase. The Repressor is a protein encoded by DNA elsewhere in the genome by a regulatory gene. It is a factor specific to the Trp operon. The repressor is an allosteric protein that has active and inactive conformations. It is inactive at synthesis and becomes activated by joining with tryptophan.Corepressor—activates the repressor (in this case the corepressor is Tryptophan)

Negative Control continuedThe repressor shuts down transcription with help of the co-repressor Trp.This is an example of negative feedback (or negative control)—because the end-product (Trp) shuts down its own production.

TrpR gene (regulatory)Trp operatorTrp operon is

“Repressible.”Normally it’s turned on, but it’s inhibited when Trp is abundant.

Lac Operon: An inducible operon

Background: Jacob & Monod (1961)Lactose = Milk sugar. It is a disaccharide of glucose and galactoseWhen you drink milk, E. coli in your gut digest lactose to obtain glucose (and galactose) for energy (ATP) production

Bond hydrolyzed by β-galactosidase

Glucose Galactose

Lac operon structureThree structural genes comprise this operon

1. β-galactosidase: hydrolyzes the lactose dimer into the 2 sugars2. Permease: A membrane protein that moves lactose into the cell3. Transacetylase – its purpose is not fully understood

lac repressor gene

Lac operon repressor

Default: The lac operon is turned on by default—but, a repressor protein is produced in active form, so it binds to the operator and shuts off the operon.The repressor binds without a corepressor. So, the lac operon is generally turned off.

Lac operon repressor, continuedWhen present, lactose binds to the repressor (technically allolactose, a lactose isomer, binds to the repressor)This deactivates the repressor, so that it can’t bind to the operatorThus, transcription is “induced” by lactose (allolactose)

Lac operon, penultimate summary

The Lac Operon is an inducible operonInduced (turned on) when lactose is abundant

The Trp Operon is a repressible operonRepressed (turned off) when Trp is abundant

Inducible operons generally involve catabolismThe operon is activated to break down a molecule

Repressible operons involve anabolism The operon is activated to build a molecule

Both are examples of negative gene regulation or negative control of genes because the operon is shut down by the active form of the repressor

Lac Operon: Also has positive gene regulation

Besides the operator, which can be blocked by a repressor to turn transcription off, the Lac operon promoter has a CRP binding site, which controls the overall rate of transcription when the operon is on. CRP = cyclic AMP Receptor Protein: a regulatory protein that enhances the binding of RNA polymerase, thus stimulating transcription. This is positive gene regulation because the operon is stimulated by an activator molecule.

Lac operon requires more than lactose to be fully activated.To be completely efficient, glucose must be depleted. Why?

activated by CRP attachment

cAMP

Lac OperonThe Lac Operon is turned on (induced) when Lactose (allolactose) deactivates its repressorThe CRP is activated by cAMP. When activated, CRP binds to DNA at the CRP binding site next to the promoter and enhances RNA polymerase binding, thus improving the efficiency of transcription.Thus, the Lac Operon is most efficient when glucose concentration is low and cAMP concentration is high.

What’s with cAMP?

cAMP = cyclic adenosine monophosphate. It is the energetically depleted relative of ATP.

High cAMP concentration signals low glucose content. Why?

Why is low glucose content important to the lac operon? When glucose is low, ATP gets used up and AMP increases in concentration. The bacterium needs to catabolized lactose to glucose for respiration to produce more ATP from the AMP.

Lac operon, final summary

When no lactose, there is no lac operon activation (the repressor is active)

When lactose and ATP are abundant, there is slight activation of the operon because the repressor is deactivated and RNA polymerase works at a low level

Cells won’t waste ATP, etc.,making enzymes to breakdown lactose when there is no lactose.

Lac operon, final summary continued

When there is lactose and lots of cAMP, that means ATP is low, and the lac operon works at peak efficiency.

Lactose binds and inactivates the repressorcAMP activates CRP which binds to the DNA. RNA polymerase then

jumps on the promoter like a duck on a June bug.

The on-off switch & volume analogy—think rheostat

Structural vs regulatory genesStructural genes code for enzymes and other proteins

Regulatory genes include the genes that produce products like regulatory proteins that bind to operators, cAMP receptor protein, and transcription and translation factors. These control the expression of structural genes.

Regulation of eukaryotic gene expression

When can gene expression be controlled?In chromatin (packing & unpacking)During transcriptionDuring and after mRNA processingDuring and after translation.

Gene Expression: Refers to the timing and circumstances in which genes do their work. Obviously, all genes can’t be turned on all the time.

Control of gene expression depends largely on genome organization.

Multicellular eukaryotes have the special problem of cellular differentiation: specialized cells (e.g., liver, heart, muscle), need to turn their genes on and off at different times. Some genes are never expressed!

Chromatin: Replication & Transcription

When tightly packed, DNA in chromatin cannot be not replicated or transcribed

Chromatin packing loosens to allow replication & transcription -Euchromatin: Loosely packed (ready for action) -Heterochromatin: Never unpacked, except for interphase replication

Chromatin Packing:-Chromatin: Complexes of DNA and protein-Histones: Proteins playing a key role in DNA

packing-Nucleosomes: “Beads” of histones wrapped with

DNA -Chromatin fiber: Coils of nucleosomes-Looped domains: chromatin fiber attached to a non-

histone scaffold-Compacted chromatin folds = visible chromatids

Understanding genome organization is the first step to understanding gene control.-Expression of genes is partly a function of how they are packed and ordered.

Introduction to genome organization

More common mechanisms for controlling gene expression

Because of cellular differentiation, only 3-5% of genes need be expressed in a given cell

How is this expression most commonly controlled?

Methods of contol include:Histone acetylation (--COCH3): Loosens histone

grip on key genesDNA methylation (--CH3): Deactivates

unnecessary genes

Control of Transcription

Most gene expression is controlled at the transcription stage.

Control of Transcription: Control ElementsTranscription Factors: Proteins that bind to DNA to help activate

or deactivate transcriptionControl Elements: DNA sites that bind transcription factorsEnhancers: Distal control elements—DNA sequences far from the

geneProximal control elements: DNA sequences close to promoter.

Control of Transcription: FactorsTranscription factors: proteins that affect timing and expression

of gene by binding to the DNA’s elementsEvery protein that facilitates or inhibits transcription is a factor.

Factors recognize elements or other factors.

Control of Transcription: Activators

Bending of DNA places these distant complexes at the promoter.

Activators are factors that bind to Enhancers (i.e., at sites distant from the promoter).

How do activator/enhancers help when they are located far away?

Control of Transcription: Other issues

Are there repressors in eukaryotes like the ones in prokaryote operons?

Yes, “silencers” can bind to control elements and block activators and other transcription factors, but eukaryotic genes are turned off by default, so mostly they need to be turned on, not off.

prokaryotic repressor

Control of TranscriptionHow do factors attach to DNA

All transcription factors share similar “DNA-binding domains”

Proteins consist of domains with specific functions. Transcription factors have domains that allow them to bind to DNA.

A generic protein

Control of Translation--mRNA processing

After transcription: pre-mRNA must be converted to mRNA in the nucleus. Control can consist of:

1. Failure or slowing of this processing

2. Alternative splicing of introns to produce different mRNAs from same pre-mRNA.

Movie

Control of Translation: mRNA DegradationIn the cytoplasm: mRNA tends to be a fragile molecule that breaks down easily

The longer it survives, the more protein it codes

Example: Hemoglobin mRNA is long-lived—this increases production of hemoglobin in red blood cells.

Chromatin changes

Transcription

RNA processing

mRNAdegradation

Translation

Protein processingand degradation

Degradation of mRNAOR

Blockage of translation

Target mRNA

miRNA

Proteincomplex

Dicer

Hydrogenbond

The micro-RNA (miRNA)precursor foldsback on itself,held togetherby hydrogenbonds.

1 An enzymecalled Dicer movesalong the double-stranded RNA, cutting it intoshorter segments.

2 One strand ofeach short double-stranded RNA isdegraded; the otherstrand (miRNA) thenassociates with acomplex of proteins.

3 The boundmiRNA can base-pairwith any targetmRNA that containsthe complementarysequence.

4 The miRNA-proteincomplex prevents geneexpression either bydegrading the targetmRNA or by blockingits translation.

5

RNA Interference: a mechanism for controlling mRNA expression. Small interfering RNAs (siRNA) or micro RNA (miRNA) are the key in

mRNA degradation

Control of translation: Stopping the act

Translation is stopped mostly at initiationTwo general ways:

1. Regulatory proteins bind to mRNA initiation site2. Translation initiation factors are inhibited, e.g., by

phosphorylation.

Post-translational ControlThis occurs in various ways: 1. Processing of peptides is slowed, stopped, or speeded up.

e.g., insulin needs to be cleaved to worke.g., hemoglobin needs its four parts assemblede.g., membrane proteins often need sugars to be attached

2. Selective degradation-analogous to mRNA degradation-ubiquitin attaches to proteins & proteasomes gobble them up

Control of eukaryotic gene expressionSummary

packaging

number of gene copies

transcriptional control(factors and elements)

post-transcriptional modification of mRNA

rate of mRNA degradation

control of translation

rate of protein degradation

Genome organization: Prokaryotes vs. Eukaryotes

Eukaryotes: 97% of DNA is not genes, does NOT code

for mRNA, tRNA, or rRNA

Repetitive DNA: non-coding, repeating sequences

Each gene controlled by a separate promoter--no operons known

Prokaryotes: Most DNA consists of genes that code for mRNA, tRNA, & rRNAGenes are grouped into operons