Lecture 6: Regulation of Transcription Continued

Question 1

When is and when isnt beta-galactosidase (LacZ) transcribed? How is this possible?

Answer 1

Only when lactose is present AND glucose IS NOT

This is because the lac operon is under the control of not only LacI (repressor) but the activator protein “Catabolite Activating Protein” (CAP) - GLUCOSE IS A LIGAND BINDING TO THE ACTIVATOR PREVENTING TRANSCRIPTION (REPRESSOR LIGAND)

Question 2

Why is there no Lac Operon expression in the absence of an activator?

Answer 2

The Lac promoter is a weak promoter

Question 3

How does RNA Polymerase interact with the CAP activator?

Answer 3

The alpha-CTD domain interacts with CAP at the CAP binding site

Question 4

What is the ligand for CAP?

Answer 4

cAMP is the ligand (NOT GLUCOSE)

But: low glucose = high cAMP vice versa

Question 5

When does CAP bind to DNA?

Answer 5

when cAMP is bound to it (therefore glucose levels low)

Question 6

What happens to CAP when the ligand cAMP binds?

Answer 6

The DNA binding domains (F-helices) rotate 60 degrees, allowing for interaction with DNA in the major grooves

Question 7

How many promoters does CAP activate?

Answer 7

> 100

Question 8

What is the molecular mass of CAP and what does it bind to as a dimer?

Answer 8

45kDa. DNA.

Question 9

How many components are required for the simplest of CAP-dependent promoters?

Answer 9

3: CAP, RNA polymerase, and DNA.

most bacterial and eukaryotic promoters are more complicated

Question 10

Can CAP bind to different sites with respsect to the promoter?

Answer 10

Yes. E.g., -41, -61, -92

Question 11

How is it CAP can bind at various different sites?

Answer 11

It can bind at multiple that enables it to be on the same helical face of DNA to RNA polymerase.

Question 12

How is it CAP can bind at various different sites?

Answer 12

It can bind at multiple that enables it to be on the same helical face of DNA to RNA polymerase.

Question 13

Explain Class 1 CAP-dependent promoters.

Answer 13

1. Class 1: only require CAP for activation and have a single site for CAP binding located upstream from the promoter. Can be at various distances but must be on the same helical face as RNA polymerase. E.g., Lac

Question 14

How many classes of CAP-dependent promoters are there?

Answer 14

3

Question 15

Explain Class 2 CAP-dependent promoters. Where does alpha-CTD bind?

Answer 15

1. Only requires CAP for activation
2. Have a single CAP site that overlaps the promoter
3. replaces -35 site
4. E.g., galP1 promoter

alpha-CTD binds on the other side of CAP compared to class 1. Still binds to AR1.

HOWEVER, there is a second interaction between AR2 and alpha-NTD

Question 16

What part of the CAP protein interacts with RNA polymerase?

Answer 16

AR1

Question 17

Explain Class 3 CAP-dependent promoters. Where does alpha-CTD bind?

Answer 17

1. Requires 2 or more CAP dimers

2. e.g., araBAD and malK

Question 18

What are the roles of RNA polymerase alpha subunit?

Answer 18

1. Not much - no regulatory functions = sigma factor controls all the targeting
2. Contacts the CAP/cAMP activator complex with alpha-CTD
3. Contacts the UP element regulating promoter strength (i.e., acting as a transcription factor)

Question 19

How does MerR activate transcription without contacting the RNA polymerase?

Answer 19

1. MerR controls gene “merT” - encodes enzyme giving cells resistance to toxic effects of mercury
2. MerR binds sequence between -10 and -35 regions of MerT promoter.
3. Mercury activates merT expression
4. MerT promoter is sigma-70; distance between -10 and -35 elements is 19bp as opposed to regular 15-17bp
5. therefore the two sequence elements recognised by the sigma factor are neither optimally separated or aligned

Question 20

What happens to MerT in the presence of the transcription factor MerR and mercury (Hg)?

Answer 20

Goes from a 19bp spacer (between -10 and -35) because the transcription factor changes conformation = twisting of DNA

the twisting of DNA moves the -10 and -35 sites to the position they should be

Question 21

Can transcription be regulated outside of transcription initiation

Answer 21

Yes - premature transcription termination

Question 22

How does premature transcription termination control the tryptophan biosynthetic operon?

Question 23

Describe the tryptophan biosynthetic operon

Answer 23

trp operon contains five structural genes: trp A, B, C, D, E

Only expressed when tryptophan is limiting

Question 24

What are the two levels of regulation for the tryptophan biosynthetic operon?

Answer 24

1. transcription repression by the Trp repressor (regulation of initiation)
2. attenuation (regulation after the initiation)

Question 25

what is the distance between the transcription and translation start point

Answer 25

It varies. It is relevant to regulation.

Question 26

What is the leader region?

Answer 26

Has regulatory function. Controls whether entire mRNA is made or just a small RNA sequence (termination at the end of the leader sequence)

It is the region in-between the transcriptional ad translational start points

Question 27

How does the leader region lead to transcription termination?

Answer 27

Formation of hairpin loop structures between regions 1,2,3, or 4