Molecular mechanism of transcription activation by E. coli RNAP holoenzyme

Question 1

Bacterial promoters are naturally ‘tuned down’. What does this leave space for?

Answer 1

Up-regulation (activation)

Question 2

What are activators?

Answer 2

Transcription factors that activate transcription in a gene-specific manner
(e.g. temperature is not an activator)

Question 3

If all bacterial promoters had consensus sigma-binding elements, what would it be like?

Answer 3

All cars going at maximum speed on the street all the time!

Question 4

True science begins when you start what?

Answer 4

Quantifying things
e.g.:
How many RNAs does RNAP make per DNA?
How much time does it take? Seconds/minutes/hours?

Question 5

How would you discover a bacterial transcription activator?

Answer 5

1. Purify RNAP holoenzyme (alpha2, beta, beta’, omega, sigma) using chromatography. Verify that it worked (e.g. by Bio-rad assay)
2. Mix RNAP holo, DNA template containing lac promoter, NTPs, and alphaP32-UTP together in multiple (e.g. 8) tubes. Incubate at 37°C, say for 30 min
3. Measure amount of radioactive RNA produced in each tube using a Geiger counter. Calculate how many RNA RNAP made in each tube. All tubes should be the same.
4. Make a new cell extract and fractionate cell lysate. Repeat transcription for each fraction
5. Quantification of RNA identifies the fraction containing the activator (a simple yes/no assay for RNA would not be enough)
6. Run the fraction on SDS-polyacrylamide gel

Question 6

How many ‘modules’ does CAP, and all activators have?

Answer 6

3:

RNAP-binding, switch, DNA-binding

Question 7

What was the first discovered activator in 1971?

Answer 7

Catabolite-activating protein (CAP)

Question 8

What genes does CAP activate, and when?

Answer 8

CAP activates ~20 genes when bacteria are starved for glucose. These genes allow bacteria to digest other carbon sources, e.g. lactose (the lac operon) and galactose

Question 9

What genes does CAP activate, and when?

Answer 9

CAP activates ~20 genes when bacteria are starved for glucose. These genes allow bacteria to digest other carbon sources, e.g. lactose (the lac operon) and galactose

Question 10

Activators help RNAP to overcome what?

Answer 10

The ‘hardest’ step

Question 11

What is the hardest step of transcription?

Answer 11

Initiation

Question 12

Which step does CAP activate, initiation or elongation?

Answer 12

Without CAP, lac promoter makes 1 RNA in ~10 minutes. The gene is 3000 nt long.
At 30 nt/second, it should take 100 sec to transcribe lac gene (1.5 min). This is much less than 10 min. What is RNAP doing for 8.5 min?

It takes time for RNAP to bind promoters and initiate transcription.
I.e. initiation is the rate-limiting step of transcription

Question 13

Which step does CAP activate, initiation or elongation?

Answer 13

Without CAP, lac promoter makes 1 RNA in ~10 minutes. The gene is 3000 nt long.
At 30 nt/second, it should take 100 sec to transcribe lac gene (1.5 min). This is much less than 10 min. What is RNAP doing for 8.5 min?

It takes time for RNAP to bind promoters and initiate transcription.
I.e. initiation is the rate-limiting step of transcription

Question 14

Which step does CAP activate, initiation or elongation?

Answer 14

Without CAP, lac promoter makes 1 RNA in ~10 minutes. The gene is 3000 nt long.
At 30 nt/second, it should take 100 sec to transcribe lac gene (1.5 min). This is much less than 10 min. What is RNAP doing for 8.5 min?

It takes time for RNAP to bind promoters and initiate transcription.
I.e. initiation is the rate-limiting step of transcription

Question 15

Only phage promoters let RNAP ‘cross’ the initiation ‘river’ in one try (it takes it

Answer 15

Because phage promoters are designed to hijack bacterial RNAP

Question 16

Do bacterial promoters have perfect or non-perfect -35 and -10 elements? And what does this mean?

Answer 16

Non-perfect
Therefore, in the absence of an activator, most RNAP promoter
binding events do not result in promoter opening.
Instead, RNAP falls off, and then has to start all over. After many failed attempts (and after ~10 min of constant trying). RNAP finally succeeds to open the promoter and to make one RNA

Question 17

In what 2 ways can CAP activate transcription?

Answer 17

1. Stabilises RNAP-promoter interactions

2. Stimulates the conversion from closed complex to open complex

Question 18

Will activators make a difference if a promoter already has a consensus (perfect) sequence?

Answer 18

No, activators won’t make any difference

Question 19

Outline how CAP stabilises RNAP-promoter contacts

Answer 19

This mechanism is also called ‘activation by recruitment’. This is a misleading term because
CAP does not bind to RNAP in solution, and does not drag RNAP to the promoter. CAP sits on the DNA and waits for RNAP to bind

Question 20

Outline how CAP stabilises RNAP-promoter contacts

Answer 20

This mechanism is also called ‘activation by recruitment’. This is a misleading term because
CAP does not bind to RNAP in solution, and does not drag RNAP to the promoter. CAP sits on the DNA and waits for RNAP to bind

Question 21

How do we know that CAP acts through the C-terminal domain of the alpha subunit?

Answer 21

Delete the C-terminal domain of the alpha subunit. In the absence of CAP, this mutant RNAP will transcribe as much RNA as wild type RNAP. However, in the presence of CAP, the mutant RNAP will not respond to the activator.

Question 22

How do we know that CAP acts through the C-terminal domain of the alpha subunit?

Answer 22

Delete the C-terminal domain of the alpha subunit. In the absence of CAP, this mutant RNAP will transcribe as much RNA as wild type RNAP. However, in the presence of CAP, the mutant RNAP will not respond to the activator.

Question 23

Do CAP and RNAP have to be on the same face of the DNA helix for activation to work?

Answer 23

Yes, as long as CAP and RNAP are on the same face, the alpha subunit linker has enough reach to allow CAP activation from 2-3 DNA turns away!

Question 24

Do CAP and RNAP have to be on the same face of the DNA helix for activation to work?

Answer 24

Yes, as long as CAP and RNAP are on the same face, the alpha subunit linker has enough reach to allow CAP activation from 2-3 DNA turns away!

Question 25

How much is DNA bent during CAP activation?

Answer 25

Almost 180 degrees

Question 26

Do natural E. coli promoters have ‘defects’ in consensus sequences?

Answer 26

Yes

Question 27

Do phage early promoters have consensus sequences?

Answer 27

Yes

Question 28

Activators up-regulate promoters that have what in consensus sequences?

Answer 28

Defects

Question 29

In what steps do activators act?

Answer 29

At the INITIATION step, at promoter binding and promoter melting steps

Question 30

What are the two rate-limiting steps of initiation?

Answer 30

Promoter binding and promoter melting

Question 31

What is the first discovered activator?

Answer 31

CAP

Question 32

What are the three modules of CAP?

Answer 32

DNA-binding
RNAP-binding
Allosteric sensor

Question 33

Where does CAP bind?

Answer 33

UPSTREAM from the transcription start site

Question 34

Does CAP bend DNA?

Answer 34

Yes

Question 35

What does CAP stabilise?

Answer 35

RNAP-DNA interactions through the alpha-subunit of RNAP

Question 36

Does CAP bind on the same or different face of the DNA helix as RNAP?

Answer 36

Binds on the same face