Lecture 6: Post-transcriptional regulation of gene expression

Question 1

Describe some eukaryotic Pol II post-transcriptional events

Answer 1

• RNA Pol II complex
• Possible attenuation → RNA transcript aborts
• 5’ capping, splicing, 3’ end processing
  
  • If these systems fail → non-functional RNA sequences
  • Degraded in the nucleus
• Anything that passes these is modified further → exported through the nuclear export into the cytosol.

Question 2

What do eukaryotic Pol II post-transcriptional events require?

Answer 2

• Factors that bind to the phosphorylated C-terminal domain (CTD) of RNA Pol II
• CTD of RNA Pol II comprises multiple repeats of a 7 amino acid sequence YSPTSPS.
• Phosphorylation of seryls 2 and 5 → transition from transcriptional initiation to elongation

Question 3

Describe the addition of a 5’ cap

Answer 3

• Nascent RNA transcript w triphosphate at the front
• RNA triphosphate nibbles it and removes one phosphate
• Provides a site for guanylyl transferase to cleave diphosphate of a GTP
• Cleaved diphosphate is then attached to remaining pair of phosphates → triphosphate linkage
• Methyltransferase that produces a 7-methylguanosine
• Methylation of the first (n sometimes second) ribose

Question 4

What are the enzymes involved in capping?

Answer 4

• RNA triphosphatase
• Guanylyl transferase
• Methyltransferase(s)
• All recruited by the phosphorylated CTD of Pol II.

Question 5

What are the roles of the 5’ mRNA cap?

Answer 5

• Distinguishes Pol II transcripts from other RNA molecules
• Stabilises the RNA - there is no 5’ phosphate, so it is resistant to 5’ exonucleases
• Aids in further processing (exon definition hypothesis, later) and export to the cytosol (see later)
• Required for efficient translation of mRNAs (see later)
• 5’ capping is co-ordinated with transcription

Question 6

What is the role of the 5’ mRNA cap in protection, and how does mRNA degradation begin?

Answer 6

• A 5’ cap protects mRNAs from exonuclease activity.
• Regulation of cellular mRNA levels therefore depends in part on mRNA decapping pathways.
• A multi-subunit cytosolic ATP-dependent decapping enzyme complex removes the 5’ cap
• Restores a 5’ phosphate on the mRNA. The mRNA can now no longer be bound by ribosomes and so can no longer be translated.
• The mRNA is now degraded by a 5’-3’ RNAse

Question 7

What did Thomas Cech discover when rDNA from Tetrahymena strain 6UM was hybridized to its transcript?

Answer 7

• rDNA from Tetrahymena strain 6UM was hybridised to the transcript of this region.
• After electron microscopy, a displaced strand of DNA (orange) was replaced by a DNA (blue)-RNA (green) hybrid and there was a loop characteristic of an intron (ivs, intervening sequence)

Question 8

What is an ivs?

Question 9

How is the intron excised from the Tetrahymena primary transcript?