Lecture 6. Post-Transcriptional Regulation of Gene Expression 1 Flashcards
What are the eukaryotic Pol II post-transcriptional events?
Possible attenuation (RNA transcript aborts)
5’ capping
Splicing
3’ end processing
Possible RNA editing
Nuclear export
What do all eukaryotic Pol II post-transcriptional events require?
Factors that bind the phosphorylated C-terminal domain (CTD) of RNA PolII
The CTD of RNA PolII comprises multiple repeats of a 7 amino acid sequence YSPTSPS Phosphorylation of seryls 2 and 5 marks the transition from transcriptional initiation to
elongation
Why is the 5’ cap put on backwards?
The 5; end of the transcript now masquerades as the 3’ end with a 3’ hydroxyl (no longer at risk of degreadtion)
How is the 5’ cap added?
Nascent RNA transcript has phosphate removed from 5’ end by RNA triphosphatase
Guanylyl transferase cleaves diphosphate from GTP and adds the G and P to the RNA transcript
Methylate (add CH₃) G to generate 7-methylguanosine and first ribose also methylated
What is the importance of the 5’ cap?
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) and export to the cytosol
And is required for efficient translation of mRNAs
What is the key principle of the 5’ cap?
5’ capping is co-ordinated with transcription
How does decapping of the 5’ cap take place?
A multi-subunit cytosolic ATP-dependent decapping enzyme complex removes the 5’ cap
It 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
What is ivs (intervening sequence)?
Intron found in the 26R rRNA gene
The intron is excised from the primary transcript and the RNA can do it by itself
How could the ivs intron be removed from the Tetrahymena nuclei?
α-amanitin fungal toxin (Pol II inhibitor) so no mRNAs could be made but rRNA genes were still transcribed
A nuclease inhibitor stabilises the RNA
And with ribonucleotides ATP, GTP, CTP and radioactive ³²P-UTP
How did Cech discover the ribozyme?
Deproteinised the rRNA by phenol (denatures proteins) extraction and/or boiled the rRNA for five minutes. The intron was still excised and still accumulated
Cloned the rDNA and made rRNA in vitro using bacterial RNA polymerase. The intron was still excised and still accumulated
Cech defined the RNA itself was catalytic, with the new principle that RNA could catalyse functions that had previously been thought of as enzyme functions (ivs = ribozyme)
How does the mechanism of introns function in the Tetrahymena?
- The intron folds. A co-factor s held in a pocket: guanosine, GMP, GDP, GTP. The 3’-OH of the co-factor is a nucleophile that attacks the phosphate at the 5’ splice site
- The 3’-OH of the upstream exon attacks the phosphate at the 3’ splice site
- The exons are fused, and the intron is ultimately degraded
Two sequential transesterifications fuse the exons and release the intron
How does the mechanism of introns function in humans?
- The intron folds and the 2’-OH of the ‘branch site’ adenosine attacks the phosphate at the 5’ splice site
- This adenosine now has three phosphodiester bonds: one is an unusual 2’, 5’ phosphodiester bond. The 3’-OH of the upstream exon attacks the phosphate at the 3’ splice site
- The exons are fused and the intron is released as a lariat
Two sequential transesterifications fuse the exons and release the intron
What controls the intron mechanism?
The spliceosome: an RNA and protein complex
What is the spliceosome and what makes up the spliceosome?
The spliceosome: an RNA and protein complex
5 small uridine-rich snRNPs (small nuclear ribonucleoproteins) U1, U2, U4, U5, U6
Each snRNP is a small nuclear RNA (snRNA) complexed with at least seven protein subunits. There are extra proteins ~200 in total
What allows the spliceosome to splice precisely?
A combination of RNA base-pairing (specificity) and protein binding (stabilisation) allows precise splicing because the splice sites and the branch site are brought close together
Where does the spliceosome recognise specific sequences at?
The 5’ splice site
The branch site and
The poly pyrimidine tract (poly Y)