Molecular mechanism of transcription activation by E. coli RNAP holoenzyme Flashcards
Bacterial promoters are naturally ‘tuned down’. What does this leave space for?
Up-regulation (activation)
What are activators?
Transcription factors that activate transcription in a gene-specific manner
(e.g. temperature is not an activator)
If all bacterial promoters had consensus sigma-binding elements, what would it be like?
All cars going at maximum speed on the street all the time!
True science begins when you start what?
Quantifying things
e.g.:
How many RNAs does RNAP make per DNA?
How much time does it take? Seconds/minutes/hours?
How would you discover a bacterial transcription activator?
- Purify RNAP holoenzyme (alpha2, beta, beta’, omega, sigma) using chromatography. Verify that it worked (e.g. by Bio-rad assay)
- 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
- 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.
- Make a new cell extract and fractionate cell lysate. Repeat transcription for each fraction
- Quantification of RNA identifies the fraction containing the activator (a simple yes/no assay for RNA would not be enough)
- Run the fraction on SDS-polyacrylamide gel
How many ‘modules’ does CAP, and all activators have?
3:
RNAP-binding, switch, DNA-binding
What was the first discovered activator in 1971?
Catabolite-activating protein (CAP)
What genes does CAP activate, and when?
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
What genes does CAP activate, and when?
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
Activators help RNAP to overcome what?
The ‘hardest’ step
What is the hardest step of transcription?
Initiation
Which step does CAP activate, initiation or elongation?
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
Which step does CAP activate, initiation or elongation?
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
Which step does CAP activate, initiation or elongation?
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
Only phage promoters let RNAP ‘cross’ the initiation ‘river’ in one try (it takes it
Because phage promoters are designed to hijack bacterial RNAP
Do bacterial promoters have perfect or non-perfect -35 and -10 elements? And what does this mean?
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
In what 2 ways can CAP activate transcription?
- Stabilises RNAP-promoter interactions
2. Stimulates the conversion from closed complex to open complex
Will activators make a difference if a promoter already has a consensus (perfect) sequence?
No, activators won’t make any difference
Outline how CAP stabilises RNAP-promoter contacts
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
Outline how CAP stabilises RNAP-promoter contacts
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
How do we know that CAP acts through the C-terminal domain of the alpha subunit?
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.
How do we know that CAP acts through the C-terminal domain of the alpha subunit?
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.
Do CAP and RNAP have to be on the same face of the DNA helix for activation to work?
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!
Do CAP and RNAP have to be on the same face of the DNA helix for activation to work?
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!
How much is DNA bent during CAP activation?
Almost 180 degrees
Do natural E. coli promoters have ‘defects’ in consensus sequences?
Yes
Do phage early promoters have consensus sequences?
Yes
Activators up-regulate promoters that have what in consensus sequences?
Defects
In what steps do activators act?
At the INITIATION step, at promoter binding and promoter melting steps
What are the two rate-limiting steps of initiation?
Promoter binding and promoter melting
What is the first discovered activator?
CAP
What are the three modules of CAP?
DNA-binding
RNAP-binding
Allosteric sensor
Where does CAP bind?
UPSTREAM from the transcription start site
Does CAP bend DNA?
Yes
What does CAP stabilise?
RNAP-DNA interactions through the alpha-subunit of RNAP
Does CAP bind on the same or different face of the DNA helix as RNAP?
Binds on the same face
