Gene Expression and RNA Processing

Question 1

What are the subunits of E. coli RNA Polymerase?

Answer 1

Alpha
Beta
Beta'
W
o70

Question 2

Alpha subunit of RNA Polymerase

Answer 2

Required for assembly of core enzyme, interacts with regulatory factors (2 subunits)

Question 3

Beta subunit of RNA Polymerase

Answer 3

Takes part in all stages of catalysis

Question 4

Beta’ subunit of RNA Polymerase

Answer 4

Binds to DNA, takes part in catalysis

Question 5

W subunit of RNA Polymerase

Answer 5

Required to restore denatured polymerase to its native form

Question 6

o70 subunit of RNA Polymerase

Answer 6

Takes part in promoter recognition

Core enzyme + o70 = holoenzyme

Question 7

Initiation of Transcription

Answer 7

• Sigma (o) subunit decreases the affinity of RNA polymerase for DNA
• Sigma subunit recognizes promoter sequence (-35, -10)
• After several nucleotides of RNA are synthesized, o falls off
• No primer required

Question 8

Elongation Reaction for RNA Transcription

Answer 8

3’OH acts as nucleophile and attacks P

- PPi released, H2O added = 2Pi

Question 9

Termination of Transcription

Answer 9

• Stemloop causes RNA polymerase to pause
• rU-dA base pairs are weak, so that RNA dissociates from the DNA template and enzyme
• Protein-dependent termination (Rho protein)

Question 10

Transcription in Prok and Euk have what in common?

Answer 10

In common:

• NTP + RNAn + H2O -> RNAn+1 + 2Pi
• 3’OH group of the growing RNA chain attacks the innermost phosphate of next NTP
• RNA is formed in the 5’-3’ direction

Question 11

Transcription in Prok vs. Euk differences

Answer 11

Prok differences:
- Transcription and translation occur at same time
- Simple control elements
- Termination signal is GC rich hairpin-poly(U) structure
- mRNA is not transported across a membrane
Euk differences:
- Transcription and translation are separated in time and space
- More complex control elements
- Termination signal is poly(A) tail
- mRNA is highly processed and transported across the nuclear envelope

Question 12

Eukaryotic cells use three RNA polymerases to synthesize RNA:

Answer 12

mRNA, rRNA, tRNA

Question 13

Cis-acting elements

Answer 13

DNA sequences that regulate expression of a gene located on the same molecule of DNA

Question 14

Where is the TATA box located?

Answer 14

-25

Question 15

Inr

Answer 15

Initiator element +1

Question 16

Enhancer

Answer 16

Can be >1kilobases from start site

Question 17

DPE

Answer 17

Downstream core promotor element +30

Question 18

CAAT box and GC box

Answer 18

-40 to -150

Question 19

Pre-Initiation Complex (PIC)

Answer 19

• Transcription factor bind cis-acting elements to recruit RNA pol II
• TATA box is recognized by TFIID by the TATA Binding Protein (TBP)
• TFIID is a dynamic protein complex

Question 20

Important stuff to know for formation of the pre-initiation complex

Answer 20

1) TFFID binds DNA and the TBP domain, until eventually binds TATA box
4) TFIIB recognizes TBP/DNA complex and RECRUITS POL II!
5) TFIIH (helicase) unwinds DNA and PHOSPHORYLATES THE C-TERMINAL DOMAIN OF POL II (TRIGGER TRANSITION TO ELONGATION!)

Question 21

Additional Transcription Factors Regulate Transcription

Answer 21

• PIC initiates transcription at low levels
• Additional TFs that bind to other sites stim high lvls of transcription (expressed in a tissue-specific manner)
• 1 TF has little influence, many are needed to form a complex that stims or suppresses transcription (Combinatorial control!)

Question 22

mRNA Processing

Answer 22

• mRNA transcript is modified by 5’ cap and poly (A) tail

- Introns are removed and exons are joined during RNA splicing

Question 23

During formation of the 5’ Cap

Answer 23

1) Removal of a phosphoryl group from 5’ triphosphate
2) Diphosphate 5’ end attacks alpha-phosphorous of GTP
3) N-7 of terminal guanine is methylated. Adjacent riboses may also be methylated by guanylmethyltransferase

Question 24

Why do mRNAs have a 5’ cap?

Answer 24

Protection from degradation

Question 25

The Poly(A) Tail

Answer 25

Cleavage signal -> cleavage by specific endonuclease -(ATP -> PPi)-> addition of tail by poly(A) polymerase (~250 As)

Question 26

How is the splice site marked?

Answer 26

1 conserved sequence before 5’ splice site, 1 conserved sequence before 3’ splice site
Conserved branch site ~in middle of intron

Question 27

While some introns “self-splice” using RNA as the catalyst, most splicing rxns are

Answer 27

mediated by the spliceosome