Lecture 5-Transcription II (Martin) Flashcards
describe the 5’ cap
- triphosphate bridge
- 7-methyl guanosine
- possibly methylation at some of the 2’ OHs that are 3’ to the cap to prevent nucleophilic attack
What are the functions of the 5’ cap (4)
- causes efficient splicing
- allows it to leave the nucleus efficiently
- allows efficient transcription
- increases the T1/2 be preventing 2’OHs from attack
Explain how the cap is added to the mRNA
- PIC phosphorylated by IIH and multiple nuclear proteins will bind to the phosphorylated sites including the Cap synthesizing protein
- This forms the cap structures then dissociates
- Cap binding Complex (CBC) then binds here and holds the mRNA here
What is special about the lariat method of self splicing?
- the adenosine makes 3 bonds at the corner of the lariat
- 2’ OH of adenosine in intron acts as the nucleophile
- A will eventually form 3 bonds at the same time
snRNPs all contain ______
RNA
What is the order of snRNP binding to the RNA?
- U1, U2
- U4/U6, U5
What is the order of release of snRNPs from the RNA?
- U1, U4
- U2, U5, U6
U1 binds where?
5’ end of intron
U2 binds where? Whats special about this?
branch point (3’ of intron) but has a mismatched A that will act as a nucleophile
What is the inactive splicesome?
- U1, U2, U4, U5, U6
What is the active splicesome?
- U2, U5, U6
Eukaryotic splicing requires ______
ATP
Eukaryotic splicing will eventually form what structure?
- lariat
Where do eukaryotic snRNPs bind?
- phosphorylated sites of the pol
Why is the location of snRNPs important?
- it allows for ordered splicing of the transcript so exons aren’t paired incorrectly
Beta thalessemia is the result of _____
- beta subunit being synthesized more slowly than the alpha subunit because of a point mutation in the branch region which causes reduced binding of U2 to the branch point and inefficient splicing
- can sometimes lead to abnormal or truncated protein
Explain how eukaryotic polymerases terminate transcription (4)
- AAUAAA sequence transcribed at end of transcript
- endonuclease cleaves transcript nearby (30bp downstream)
- polyadenylate pol (ATP requiring) makes the polyA tail
- polyA tail wraps around polyA binding protein to leave nucleus
What are the functions of PolyA binding protein (3)
- helps it leave the nucleus
- improves translation efficacy in cytoplasm because it associates with 5’ cap
- protects from nucleases
What happens if you shorten the polyA tail?
- you make the RNA more susceptible to nucleases and therefore shorten the half life
How can the cell regulate the amount of protein in a cell through the poly A tail?
- if a gene has two polyA sites then the cell controls which site it uses
- at the 3’ unTRANSLATED end of the transcript are factors that increase the stability of it and therefore give it a longer half life
- if you splice at the earlier site then you decrease the amount of mRNA in the cell
Whats an example of something that uses various polyA splice sites to regulate protein concentration?
- calcintonin
- CGRP (Calcitonin gene related protein)
Explain the difference between HPV infected cells and tumorigenic HPV infected cells.
- HPV infected cells have not integrated the viral DNA into their genome so the transcripts have short T1/2s and fewer proteins made
- oncogenic virus is incorporated into the chromosome, loses the 3’ end of the transcript and thus is more stable
Explain the synthesis of rRNAs (3)
- all on one transcript
- after trasncription they are methylated on 2’OH groups to prevent nucleophilic attack
- cleaved
RnaseP
cuts 5’ end of tRNA
RnaseD
cuts 3’ end of tRNA where CCA sequence will be added
The D arm is rich in _____
- dihydrouridine
T arm is rich in ______
- ribothymidine
- pseudouridine
