1 - RNA Pol II CTD and Co-transcriptional RNA Processing - Wilson Flashcards
Draw a simple diagram showing gene expression - from DNA to protein. state why it is important that each step is coupled to the next
- allows high fidelity and efficiency of mRNA production and translation in the cytoplasm
draw a diagram showing how the 5’ end of every mRNA is capped
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why is mRNA capped?
- cap allows efficient export and translation of the mRNA in the cytoplasm
- protects against nucleases eg XrnII that will degrade the mRNA in the nucleus
when does mRNA capping occur?
co-trancriptionally, within the first 20-30 nucleotides being synthesised
draw a diagram and explain each stage of the splicing mechanism
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draw a diagram highlighting the overall stages for the processing of the 3’ end. include the structures that direct this processing
325 - 1 word
what are the functions of the polyA tail for mRNA?
protects the mRNA from 3’-5’ exonucleases (eg exosome)
how long is the pA sequence roughly?
around 50 nucleotides (this is the -AAUAAA sequence upstream of CA cleavage site
draw a diagram and explain it to show the protein complexes that aid in the 3’ end processing and addition of polyA tail
CLEAVAGE;
- CPSF (cleavage and polyadenylation stimulatory factor) recognises pA sequence and recruits CstF (cleavage stimulatory factor). this stimulates cleaveage of the RNA
- the symplekin scaffold protein brings all of the proteins together for this cleavage reaction eg polII, CPSF, CstF
POLYADENYLATION;
- PAP (polyA polymerase) recruited and adds of As to the end
- the As recognised by PABP (polyA binding protein) this aids in mRNA translation once reached cytoplasm
which form of mRNA (pre/normal) does the 3’ end processing occur?
pre-mRNA 3’ end processing
why is it necessary to define where exons are?
how is this achieved?
- necessary because exons around 200 nucleotides long whereas introns can range from 10-10^5 nucleotides long
- needle in a haystack
- interplay between proteins that bind exotic sequences and proteins that bind introns required for correct splicing
what pre-mRNA processing events are co-transcriptional? give the general reason why this happens
all of them;
- capping (5’)
- splicing
- cleavage and polyadenylation (3’)
- because the CTD of RNAp II is differentially phosphorylated and proteins required for these processing events bind to the CTD of polII. P of the CTD governs its interactions with various proteins eg Ser5 -> spliceosome assembly
describe the pol II CTD in humans
- CTD unique to pol II and is part of the Rpb1 subunit
- humans, consists of 52 repeats of the heptad sequence YSPTSPS
how does the CTD of pol II in humans differ to that of budding yeast?
- in yeast contains 26 repeat sequences not 52.
- number repeats roughly correlates to the complexity of the organism
what does the experiment shown in Q15 325 - 1 word show?
- shows that the CTD is essential for splicing from DNA templates
- once we remove the CTD then we do not get any pre-mRNA splicing (no band at the bottom of the gel)
what does the experiment shown in Q156325 - 1 word show?
- CTD is essential for 3’ end processing, more specifically cleavage
what does the experiment shown in Q17 325 - 1 word show? DRB = kinase inhibitor
- kinases required for the P of various residues on the CTD of pol II
- as DRB conc increases, less and less processing events
- P of the CTD therefore is required for BOTH pre-mRNA splicing and 3’ end processing and cleavage
- overal; inhibits co-transcriptional processes
name the CTD modifications of the individual residues within the repeats and state their functions. also state the kinases that P these residues (the ones that are known)
Y - can be P - kinase unknown
S - P during escape from initiation and the overall elongation (Cdk12)
P - Cis/trans conformational change
T - histone mRNA processing
S - initiation, RNA capping (Cdk 7) and splicing
P -Cis/trans conformational change
S - widely P, unclear function (Cdk7)
draw a graph and include a few examples of the CTD differential Phosphorylation at different timepoints
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which kinase is recruited and which residue of the CTD is phosphorylated for capping?
ser5 is phosphorylated
by Cdk7
draw a diagram of the capping process and the CTD
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describe CTD assembly of the spliceosome
phosphorylation of CTD recruits the U2AF
- U1snRNP bound to pre-mRNA
- U2AF recruits the PRP19C complex allowing assembly of the spliceosome
- subsequent splicing processes
what kinases and CTD residue is invovled in 3’ end processing ?
Cdk12 phosphorylates Ser2 of the CTD
draw a diagram of 3’ end processing and the CTD and explain it
- upstream of pA, Ser2P present at low levels
- CPSF accompanies Pol II
- transcription of pA site induces pol II pausing aided by CPSF
- triggers Ser2P by Cdk12
- CstF recruited and Cleavage and polyA take place efficiently
- the slowing down of the pol II allows efficent processing events eg cleavage
