Prokaryotic Gene Regulation

Structural gene

produces enzyme/protein that is the target of regulation

Regulatory gene

makes a product (RNA or protein) that influences the expression of the structural gene

Regulatory element

specific sequence in RNA/DNA located in the structural gene and acts as a binding site.

allows the product of the regulatory gene (the regulatory protein or RNA) to exert control over the structural gene’s expression. this is the cis-acting element.

cis-acting elements only function when attached to the target gene being regulated.

trans-actor factor: diffusable factor that is encoded elsewhere in the genome. needs to find target DNA or RNA sequence to bind to and exert its action.

e.g., transcription factors

Transcription factor

DNA binding protein that regular transcription from a promoter.

bind to sequences associated with a promoter and can influence the rate of transcription from that promoter.

Operons

groups of genes that serve a common pathway and are coordinately expressed.

this coordinated expression is achieved because the genes in an operon are co-transcribed on one mRNA molecule and co-regulated by the same regulatory elements.

alternate sigma subunits

made under different stress conditions and serve to lead RNA polymerase to specific promoters (-10 and -35 sequences) for genes needed under those particular particular stress conditions.

Regulation classification

nature of regulatory protein (repressor vs activator) and the role of the signal molecule

Negative control

regulatory protein acts as a repressor of transcription. turns off a nearby promoter when it binds to DNA

can be inducible: a modulator molecule turns ON gene expression. presence of the signal molecule makes the repressor inactive, causing it to unbind DNA and allowing transcription.

can be repressible: modulator molecule turns OFF gene expression. presence of the signal molecule makes the repressor active.

Positive control

regulatory protein = activator of transcription

inducible: turns on gene expression. presence of signal molecule makes activator active, causing it to bind DNA and stimulate transcription

repressible: turns off gene expression. signal molecule makes activator inactive, causing it to unbind DNA and stop transcription.

Lactose (lac) ope ron

lacZ, lacY, and lacA

negative inducible regulation by lac repressor (encoded by lacI gene)

• lacI is always made

• in absence of lactose, the Lac Repressor binds to a regulatory element called the operator. the binding shuts off transcription of the lac operon.

• presence of lactose: small amount of operon activity occurs, leading to the production of allolactose. allolactose binds to the lac repressor. this binding causes it to unbind from DNA. so transcription can occur.

lac operator (O) is binding site

lac repressor (I) is the protein

lac Z (structural gene)

Oc operator mutation means the sequence does not bind the lac repressor

I- repressor mutation means the repressor protein does not bind DNA.

IS mutation mean the repressor does not bind allolactose, so it does not de-repress the operon even in high lactose.

O acts in cis to Z (meaning it must be on the same DNA molecule as the structural gene).

I acts in trans to Z (repressor protein can be produced from a gene elsewhere and diffuse to bind the operator)

Oc mutation is epistatic to Is

Catabolite Activator Protein (CAP)

E. coli prefers glucose when it’s present over lactose. cell wants to repress the lac operon when glucose is present, even if lactose is also present.

Positive repressible regulation!

CAP is transcriptional activator that stimulates transcription when bound to the promoter region.

regulated by glucose leves. high glucose → low cyclic AMP (cAMP). low glucose → high cAMP

cAMP binds to CAP. causes CAP to bind to DNA and activate the lac promoter.

with respect to glucose, CAP is a repressible activator.

lac operon turned ON only when lactose is present (inactivating the repressor) AND glucose is asbent (allowing CAP-cAMP to bind and activate).

Trp operon

contains genes for tryptophan biosynthesis. This operon needs to turn off if tryptophan is present (the cell doesn’t need to make it) and turn on if tryptophan is absent.

Negative repressible regulation controlled by the Trp repressor.

• in the absence of tryptophan, the trp repressor protein is made but is inactive and doe snot bind DNA. transcription occurs.

• in the presence of tryptophan, tryptophan binds to the repressor → causes repressor to become active and bind to the operator DNA.

Trp attenuation

regulation based on the structure of the mRNA in the 5’ end of the operon

cis-element

mRNA leader sequence has 4 regions (1,2,3,4) that can fold and form diff base-pairing structures. region 4 follower by poly U segment

folding of RNA depends upon the speed of translation by the ribosome, which in turn depends on the availability of tryptophan. Region 1 of the mRNA leader sequence contains tryptophan codons.

tryptophan high → ribosomes moves quickly through the tryptophan codons in region 1. cover regions 1 and 2. allows regions 3 and 4 to pair. the 3-4 pairing forms a terminator structure, causes mRNA transcription to terminate at the polyU segment before reaching structural genes (starting at ATG).

tryptophan low → ribosomes stall at the trp codons in region 1 bc trp-tRNAs are scarce. stalling allows region 2 to pair with region 3. the 2-3 pairing prevents the formation of the 3-4 terminator loop. with the terminator loop prevented, transcription continues past the ATG and through the structural genes.