RNA biology lecture 2

Question 1

Capping overview

Answer 1

• Occurs very early elongation just after RNA Pol dis-engaged from promoter
• Cdk7 phosph Pol II Ser 5, Pol II disengages from PIC
• Transcription machinery reconfigured

Question 2

Stage II - arrest of RNAPII

Answer 2

• RNAPII stops synthesis after 20-40 bp of RNA
• RNA exits Pol II via exit channel close to CTD
• RNA Pol assoc w. processing factors, DSIF engages Pol by Spt5 interacting w/ Pol near exit channel
• Complex recognised by NELF → RNA pol arrest
• Capping E recruited to CTD

Question 3

Capping

Answer 3

• Starts after 20-30 nucleotides
• Involves 3 reactions, finalised by assoc. of modified cap nucleotide
• 1. (RNA triphosphatase carries out 5’ triphosphate → diphosphate)
• 2. (guanyltransferase transfers GTP to GMP w/ diphosphate, GMP attached to 1st nucleotide)
• 3. (methytransferase uses 5-adenosylmethionie to methylate G at pos 7)

Question 4

Capping control

Answer 4

• DXO recognises incompletely capped transcript, removes cap + destroys transcript
• Also if methyltransferase x recognise G = substrate for DXO
• If mRNA x bind cap binding complex, exposed ends recognised by decapping E Dep1.2
• Competition btw cap binding complex + DXO or Xin2 binding to cap

Question 5

Capping function

Answer 5

• Protects mRNA from degradation by 5’-3’ exonuc
• Stimulates splicing
• Nuclear-cytoplasmic export
• Translation initiation

Question 6

After capping

Answer 6

• RNA pol = arrested state, needs to be released

- Cdk9 phosph ser 2 of CTD + subunits DSIF + NELF → rearrangement of transcription complex, TF recruited, RNAP released

Question 7

Promoter proximal stalling importance

Answer 7

• Important for capping, allows gene regulation
• Regulation relies on PTEfb, recruited after pausing, causes phosph of Ser CTD
• Chance to activate genes ↑ rapidly

Question 8

pTEfb activation ↑ regulated

Answer 8

• Promoter proximal release integrated into signalling pathway
• Recruitment of pTEfb to Pol = RLS
• Activation of pTEFb = subject to ↑ controls
• Most pTEFb = initially inactive, associate w/ HEX1M1, 7SK associates + traps pTEFb inactive
• pTEFb = released due to signals like HIFa, TF, mediator

Question 9

Regulation of gene expression during drosophila development

Answer 9

• Embryogenesis, pol II recruited + subsequently paused, genes in permissive state
• Poised Pol II = present across ↑ tissues during pattern formation
• Pause Pol allows activation e.g. upon exposure of cells to gradient of morphogens