24th Feb - The First Steps of splicing Flashcards
What is UAP56?
A helicase
What are the components of complex E?
U1 snRNP Branchpoint binding protein U2f65 UAP56 U2f35 SR protein
What helps U2F65 and U2F35 bind?
The polypyrimidine tract next to AG (3’ss)
What is complex E?
A theoretical model is it was captured without ATP
Why is complex E not a great model?
Unsure whether the complex is dependent on the lack of ATP or ATP just slows down the process allowing it to be visualised
U2 binds in a non-stochastic function
What is the purpose of an Fe BABE experiment?
To determine whether two sites are close
Outline an Fe BABE experiment for determining whether the RNA is bent during splicing
Generate short lived oxygen radicals from tethered Fe3+ which break nearby RNA strands (Within 2nm). If this is attached to one end of the intron, we can identify whether the RNA is bent, as if the other end of the intron is damaged they must be bent.
YES it was damaged therefore the 5’ss and 3’ss are close , thus must be bridged somehow
What is complex I
A variant of complex E in which we prevent protein dephosphorylation
Summarise the main components of complex A
Complex A proteins
U1 snRNA
U2 snRNA
What recognises the branch site?
U2 by base pairing
Outline U2 recognition of the branch site in yeast
Base pairing displaces the branchpoint A from the new helix. In yeast an altered bp –> sequence is defective, which can only be compensated when U2 is also mutated
How does stable binding to the branchpoint occur in mammals?
Stable binding of U2 requires displacement of SF1/mBBP by UAP56 and remodelling of the U2 snRNA by Prp5, both ATP dependent helicases
What is the role of SF3A1 in complex A?
It interacts with the last stem loop of U1
What is the role of U2F65 in complex A?
It recruits SF3B1 and therefore the U2snRNP
What requires ATP during splicing?
Phosphorylation of proteins
RNA dependent ATPases
