Transcription Initiation and Elongation

Question 1

What is the catalytic site of RNA Polymerase? What happens at this site?

Answer 1

The site in which the template strands enters.
o mRNA and DNA hybrid
o The coding strand is sequestered away from the catalytic site

Question 2

What is the winding point of RNA Polymerase? What happens at this site?

Answer 2

o Re-annealing of DNA

Question 3

What enzyme is responsible for the unwinding of DNA?

Answer 3

Helicase

Question 4

What enzyme is responsible for the re-annealing of DNA?

Answer 4

Topoisomerase

Question 5

What creates the transcriptional bubble? How does it move?

Answer 5

• DNA is melted (helix dissociated)
o The bases are unpaired, creating the bubble
• The bubble moves along the strand by Pol movement

Question 6

What is the unwinding point? What happens at this point?

Answer 6

• Unwinding point: at the edge of Pol

o Separation of coding and template strands

Question 7

What is the main component of RNA polymerase?

Answer 7

Holoenzyme

Question 8

Which types are RNA are synthesized by RNA polymerase?

Answer 8

• All RNA is synthesized by the same Pol

o mRNA, tRNA, rRNA

Question 9

What is the holoenzyme?

Answer 9

about 460 kDA
o	Initiation complex
o	Has 2 functional components
1.	Core enzyme
2.	σ subunit (σ factor)

Question 10

What is the core enzyme?

Answer 10

o	About 400 kDa
o	Made of multiple subunits: α2ββ’ω
o	Involved in all stages of transcription
o	α 
1.	Required for core assembly
2.	CTD: C-terminal domain
3.	Binding interactions with DNA and regulatory proteins
o	β and β’: catalytic site
o	ω: core assembly

Question 11

What is the role of the β and β’ subunits of the core enzyme?

Answer 11

Catalytic site

Question 12

What is the role of the ω subunit of the core enzyme?

Answer 12

Core assembly

Question 13

What is the σ factor?

Answer 13

o Promoter recognition and binding

o More variable than core enzyme

Question 14

What happens if RNA Pol doesn’t bind the promoter?

Answer 14

Non-sensical RNA is synthesized

Question 15

What are the features of RNA Pol and DNA for transcription initiation to occur?

Answer 15

RNA Pol needs to precisely recognize and bind only the promoter sequence
o Requires high binding affinity
o Very limited DNA sequence

Question 16

What are the features of RNA Pol and DNA for transcription elongation to occur?

Answer 16

o Elongation requires low binding affinity to allow for constant binding and release
o Needs to recognize all DNA sequences

Question 17

What is the holoenzyme dilemma?

Answer 17

Initiation:
- High binding affinity
- Need to recognize very limited DNA sequence
Elongation
- Low binding affinity
- Need to recognize all DNA sequences

Question 18

What is the solution for the holoenzyme dilemma?

Answer 18

reversible association of the σ factor with the core enzyme
o Core enzyme is responsible for elongation
 Too generalized for specific promoter recognition
o σ factor has a high binding affinity for promoter
 Only recognizes promoter sequence
 σ binds to promoter only after binding the core
 α subunit of the core enzyme facilitates promoter recognition

Question 19

What is the closed binary complex?

Answer 19

σ bound but DNA wound

o Holoenzyme + DNA

Question 20

What is the open binary complex?

Answer 20

σ bound and DNA has unwound

o Holoenzyme + DNA

Question 21

What is the ternary complex?

Answer 21

There is RNA (aborted transcripts)
o As long as σ is bound to the core, the core cannot move forward
o As RNA is synthesized, it is aborted, until σ is released
o Holoenzyme + DNA + RNA

Question 22

What is the ternary elongation complex?

Answer 22

Core, DNA, RNA

Question 23

When does initiation end?

Answer 23

As σ is released