27th Feb - B--> Bact --> C--> C* and Post-splicing Flashcards
How is complex E accumulated for analysis?
Incubate without ATP
How is complex A accumulated for analysis?
Add anacardic acid or 2’-O-methyl oligonucleotide complementary to U6
How is complex B accumulated for analysis?
Limit the time of incubation
How is complex Bact accumulated for analysis?
Use pre-mRNA with a very short polypyrimidine tract
How is complex C accumulated for analysis?
Use pre-mRNA lacking a 3’ exon
How are splicing complexes purified for analysis?
Using an antibody specific to the complex of an MS2-MBP affinity purification
How was the mammalian tri-snRNP U4/U6.U5 discovered?
Applied psoralin and irradiated with 365nm light causing crosslinking
What is the only splicing complex with all 5 snRNAs?
Complex B
Describe complex B
U1 remains base paired to the 5’ss and U2 to the branch site, and U6 to U4 but the 3’ end of U6 is base paired to U2
What drives the complex B –> Bact transition?
Helicases
Prp* hydrolyses ATP and displaces U1
Brr2 progresses down the ss portion of U4, down to the base paired region and opens it up, seperating U4 from U6 therefore U4 leaves
Describe complex Bact
Rearrangement of U6 pushes off U1
A U2-U6 active site is present
Branch point is identified but held away to prevent splicing
What is the complex B*?
The active complex
What is the C complex?
The final complex after reaction has happened for 5’ss, so the branch site is in the active site
Describe the C complex
The branch site leaves the active site leaving it free for the 3’ss
How is the branch site removed from the active site?
There is a hinge between U2 and the branch site helix which rotates to pull the branch site out of the active site
