Prokaryotic Gene Regulation

Question 1

Where does gene expression occur?

Answer 1

There is no nucleus to separate processes so transcription and translation are coupled.

Question 2

Describe the gene architecture in prokaryotes.

Answer 2

• 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

Question 3

What is an operon comprised of?

Answer 3

A promoter sequence, followed by an operator, followed by several structural genes.

Question 4

How are operons controlled?

Answer 4

By regulatory genes found elsewhere on the chromosome which regulates the expression of the structural genes in response to an environmental change.

Question 5

What is σs used for?

Answer 5

Needed in starvation or hyperosmolarity- can put cells in stationary phase.

Question 6

What is σ 32 used for?

Answer 6

Needed during heat shock and nutrient starvation. Produces heat shock proteins which prevent protein unfolding at high temperatures.

Question 7

How are the -35/-10 sites different in σ 32?

Answer 7

They are slightly closer together than in σ 70 and σs.

Question 8

What is σ 54 used for?

Answer 8

Needed during nitrogen and nutrient starvation.

Question 9

What are the binding sites of σ 54 to the promoter?

Answer 9

-24/-12

Question 10

What does σ 54 need to initiate transcription?

Answer 10

ATP and an enhancer protein to bend the DNA into a loop.

Question 11

What is σ 70 used for?

Answer 11

Control of the expression of most genes needed to survive.

Question 12

What are the genes encoded for by the arabinose operon?

Answer 12

AraA, AraB, AraD

Question 13

What are the cis elements in the arabinose operon?

Answer 13

AraO1, AraO2, AraI binding site, CAP binding site.

Question 14

What are the trans elements in the arabinose operon?

Answer 14

AraC, cAMP and CAP.

Question 15

Describe the negative regulation of the arabinose operon.

Answer 15

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.

Question 16

Describe the positive regulation of the arabinose operon.

Answer 16

If arabinose is present, AraC binds to AraI1 and AraI2, meaning there is no DNA looping, allowing promoter access and transcription.

Question 17

Describe the role of catabolite repression in the arabinose operon.

Answer 17

If glucose is absent, adenylate cyclase activity increases, cAMP binds to CAP, activating transcription. Positive regulation.

Question 18

Describe the autoregulation of the arabinose operon.

Answer 18

If there are high levels of AraC, AraC binds to AraO1. RNAP can’t access Parac promoter, meaning there is no transcription of AraC.

Question 19

Why is the autoregulation of the arabinose operon necessary?

Answer 19

To regulate the level of positive/negative regulation of the operon.

Question 20

What does the tryptophan operon consist of?

Answer 20

Transcription regulatory region and 5 structural genes.

Question 21

Describe how the trp operon can be repressed by a repressor.

Answer 21

TrpR produces the repressor protein. At high levels of trp, trp binds to the repressor which can then bind to the operator- blocks transcription.

Question 22

To what extent does the repressor repress the trp operon?

Answer 22

70-fold repression.

Question 23

What is attenuation?

Answer 23

Post-transcriptional control of gene regulation, involving the formation of two different stem loops in the leader mRNA (trpL).

Question 24

To what extent does attenuation repress the trp operon?

Answer 24

An additional 10-fold repression. (Overall repression is 700-fold)

Question 25

Why is attenuation only possible in prokaryotes?

Answer 25

Transcription and translation are only coupled in prokaryotes.

Question 26

Give an example of another operon where attenuation occurs.

Answer 26

His operon.

Question 27

What happens when [trp] is low?

Answer 27

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.

Question 28

Why is the 2/3 stem loop not an intrinsic terminator?

Answer 28

Stem loop is followed by Gs/Cs and is not a U-rich region (would release transcript due to weak A-U bonds).

Question 29

What happens when [trp] is high?

Answer 29

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.

Question 30

What is lacI?

Answer 30

A separate transcript that encodes a lac repressor protein.

Question 31

Describe the structure of the lac repressor protein?

Answer 31

Multimeric protein of 4 subunits. 3° structure is a helix-turn-helix motif so it can bind to major groove of DNA.

Question 32

What does the lac repressor protein do?

Answer 32

Binds to the lac operator (O1 and O2 sites) to inhibit transcription.

Question 33

When can the lac repressor protein not bind to the lac operator?

Answer 33

If it is bound to an inducer.

Question 34

Describe the lac operator.

Answer 34

An imperfect inverted repeat region of DNA that lies partly in the promoter region. Consists of O1, O2 and O3.

Question 35

What happens when lactose is absent?

Answer 35

LacI binds to the operator. No lac genes are expressed.

Question 36

What happens when lactose is present?

Answer 36

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.

Question 37

Describe the feedback control mechanism of the lac operon.

Answer 37

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.

Question 38

Describe the role of catabolite repression in the regulation of the lac operon.

Answer 38

If glucose is absent, adenylate cyclase activity increases and more cAMP is produced. cAMP binds to CAP, activating transcription. Positive regulation.

Question 39

When does maximal transcription of the lac operon occur?

Answer 39

- glucose is absent - lactose is present - there has been interaction of CAP and cAMP - allolactose binds to lacI and the repressor is released

Question 40

What is the role of cAMP in catabolite repression?

Answer 40

- 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

Question 41

How does CAP interact with operons?

Answer 41

- binds upstream of the promoter - increases the affinity of RNAP for the DNA - bends DNA to increase promoter accessibility

Question 42

What happens to the lac/ara operon if glucose is present?

Answer 42

The structural genes are not transcribed.

Question 43

What protein motif does CAP adopt and how does this allow CAP to bind to DNA?

Answer 43

Helix-turn-helix, binding to the major groove of DNA.