Prokaryotic Gene Regulation Flashcards
Where does gene expression occur?
There is no nucleus to separate processes so transcription and translation are coupled.
Describe the gene architecture in prokaryotes.
-have operons- mRNAs often polycistronic- not a single promoter for every gene- separate ribosome binding sites between each gene so that each gene is translated separately
What is an operon comprised of?
A promoter sequence, followed by an operator, followed by several structural genes.
How are operons controlled?
By regulatory genes found elsewhere on the chromosome which regulates the expression of the structural genes in response to an environmental change.
What is σs used for?
Needed in starvation or hyperosmolarity- can put cells in stationary phase.
What is σ 32 used for?
Needed during heat shock and nutrient starvation. Produces heat shock proteins which prevent protein unfolding at high temperatures.
How are the -35/-10 sites different in σ 32?
They are slightly closer together than in σ 70 and σs.
What is σ 54 used for?
Needed during nitrogen and nutrient starvation.
What are the binding sites of σ 54 to the promoter?
-24/-12
What does σ 54 need to initiate transcription?
ATP and an enhancer protein to bend the DNA into a loop.
What is σ 70 used for?
Control of the expression of most genes needed to survive.
What are the genes encoded for by the arabinose operon?
AraA, AraB, AraD
What are the cis elements in the arabinose operon?
AraO1, AraO2, AraI binding site, CAP binding site.
What are the trans elements in the arabinose operon?
AraC, cAMP and CAP.
Describe the negative regulation of the arabinose operon.
If arabinose is absent, AraC binds to AraO2 and AraI, forming a DNA loop ahead of the promoter, meaning that RNAP can’t access the promoter and there is no transcription.
Describe the positive regulation of the arabinose operon.
If arabinose is present, AraC binds to AraI1 and AraI2, meaning there is no DNA looping, allowing promoter access and transcription.
Describe the role of catabolite repression in the arabinose operon.
If glucose is absent, adenylate cyclase activity increases, cAMP binds to CAP, activating transcription. Positive regulation.
Describe the autoregulation of the arabinose operon.
If there are high levels of AraC, AraC binds to AraO1. RNAP can’t access Parac promoter, meaning there is no transcription of AraC.
Why is the autoregulation of the arabinose operon necessary?
To regulate the level of positive/negative regulation of the operon.
What does the tryptophan operon consist of?
Transcription regulatory region and 5 structural genes.
Describe how the trp operon can be repressed by a repressor.
TrpR produces the repressor protein. At high levels of trp, trp binds to the repressor which can then bind to the operator- blocks transcription.
To what extent does the repressor repress the trp operon?
70-fold repression.
What is attenuation?
Post-transcriptional control of gene regulation, involving the formation of two different stem loops in the leader mRNA (trpL).
To what extent does attenuation repress the trp operon?
An additional 10-fold repression. (Overall repression is 700-fold)
Why is attenuation only possible in prokaryotes?
Transcription and translation are only coupled in prokaryotes.
Give an example of another operon where attenuation occurs.
His operon.
What happens when [trp] is low?
Ribosome stalls at 2 trp codons in region 1 as charged tRNA(trp) is in short supply. Stem loop forms between regions 2+3- does not affect transcription. Region 3 is not available to form a stem loop with region 4. Get transcription of all genes and trp production.
Why is the 2/3 stem loop not an intrinsic terminator?
Stem loop is followed by Gs/Cs and is not a U-rich region (would release transcript due to weak A-U bonds).
What happens when [trp] is high?
Ribosome quickly translates region 1, covering region 2. Region 2 can’t bind to region 3, leaves region 3 available form a stem loop with region 4- terminating transcription. No transcription, no trp production.
What is lacI?
A separate transcript that encodes a lac repressor protein.
Describe the structure of the lac repressor protein?
Multimeric protein of 4 subunits. 3° structure is a helix-turn-helix motif so it can bind to major groove of DNA.
What does the lac repressor protein do?
Binds to the lac operator (O1 and O2 sites) to inhibit transcription.
When can the lac repressor protein not bind to the lac operator?
If it is bound to an inducer.
Describe the lac operator.
An imperfect inverted repeat region of DNA that lies partly in the promoter region. Consists of O1, O2 and O3.
What happens when lactose is absent?
LacI binds to the operator. No lac genes are expressed.
What happens when lactose is present?
Small amounts of allolactose are formed and acts as an inducer. Allolactose binds to the lac repressor and prevents it from binding to the operator. RNAP binds to promoter- genes are transcribed.
Describe the feedback control mechanism of the lac operon.
Production of lacZ/lacY/lacA- break down lactose and allolactose, causing the eventual release of the repressor as lactose is absent. Stops additional lac mRNA synthesis.
Describe the role of catabolite repression in the regulation of the lac operon.
If glucose is absent, adenylate cyclase activity increases and more cAMP is produced. cAMP binds to CAP, activating transcription. Positive regulation.
When does maximal transcription of the lac operon occur?
- glucose is absent - lactose is present - there has been interaction of CAP and cAMP- allolactose binds to lacI and the repressor is released
What is the role of cAMP in catabolite repression?
- cAMP acts as a positive regulator- binds to CAP and causes a conformational change in CAP that allows CAP/cAMP to bind to the operon as a dimer
How does CAP interact with operons?
- binds upstream of the promoter- increases the affinity of RNAP for the DNA- bends DNA to increase promoter accessibility
What happens to the lac/ara operon if glucose is present?
The structural genes are not transcribed.
What protein motif does CAP adopt and how does this allow CAP to bind to DNA?
Helix-turn-helix, binding to the major groove of DNA.
