RNAP

Question 1

How conserved is the process of transcription?

Answer 1

Pretty conserved within domains, and sometimes between domains. The overarching process is quite similar even if the specific details aren’t conserved

Question 2

What are 2 prokaryotic model organisms to study gene regulation?

Answer 2

E coli and B subtilis

Question 3

What are 4 eukaryotic model organisms to study gene regulation?

Answer 3

Mice, yeast, Drosophila, human cell lines

Question 4

What is RNA polymerase?

Answer 4

Enzymes that synthesize RNA from a DNA template using the process of transcription

Question 5

What 3 things were established about RNAP in early studies from the 1960s?

Answer 5

1. The basic structure
2. Transcription wasn’t random, and only happened at certain locations in the genome
3. Transcription was asymmetrical and only one strand of DNA was transcribed

Question 6

What was the basic structure of RNAP that was established in early studies?

Answer 6

1. Multiple subunits
2. The prokaryotic holoenzyme includes 2 alpha, 2 beta, one sigma, and one omega subunit
3. Overall structure of the holoenzyme is highly conserved between domains

Question 7

What is the RNAP core?

Answer 7

The alpha and beta subunits of RNAP

Question 8

What does the RNAP core do?

Answer 8

Catalytically active and uses a DNA template to make RNA

Question 9

Can the RNAP core bind to DNA and initiate transcription on its own?

Answer 9

No, it can’t bind to intact dsDNA on its own and transcribe it. But it can if there are single strand nicks in the DNA

Question 10

Why is the RNAP holoenzyme able to transcribe dsDNA when the RNAP core can’t?

Answer 10

It needs the sigma subunit to bind to and recognize promoters, then the core can initiate transcription

Question 11

What does the sigma subunit do when its on its own?

Answer 11

Nothing. It has no enzymatic activity and can’t bind to DNA on its own. It can only do so when bound to the RNAP core to form the holoenzyme

Question 12

Which RNAP subunit is the only one that isn’t absolutely essential to cell survival and viability?

Answer 12

Omega

Question 13

What does the omega subunit do?

Answer 13

Helps the beta subunit with folding and associating with the rest of the core. Prevents the beta from aggregating. Also involved in gene regulation for stress responses

Question 14

Why is the structure of the RNAP holoenzyme so stable?

Answer 14

Each subunit is making direct contacts with each other

Question 15

Which subunit in the RNAP holoenzyme makes all sequence specific contacts?

Answer 15

Sigma

Question 16

How does the sigma subunit associate with the RNAP core?

Answer 16

It’s spread out across the upstream face of the “crab claw” structure, then loops into the active site and out through the RNA exit channel

Question 17

What are the two key functions of the sigma subunit?

Answer 17

1. Direct the RNAP core to the transcription start site by sequence specific binding to the promoter
2. Support DNA melting and strand separation at the promoter