Fundamentals of Splicing - Stryer Flashcards
1
Q
Why must splicing location be extremely sensitive?
A
Even a 1nt shift could alter the ORF after ss’ –> different protein
2
Q
What is the consensus sequence at the 5’ss for vertebrates?
A
AGGUAAGU
3
Q
What is the consensus sequence at the 3’ss for vertebrates?
A
Stretch of 10 pyrimidines at the 3’ss followed by any base then C then the invariant AG
4
Q
Outline the 2 sequential transesterification reactions of splicing
A
- Cleavage of the phosophodiester bond between the upstream exon and the 5’ end of the intron, through attack from the 2’OH on the branchpoint A.
- The 3’OH terminus of exon 1 attacks the phosphodiester bond between the intron and exon 2. Exons 1 and 2 become joined and the intron is released in lariat form
5
Q
Outline the process of splicing
A
- U1 snRNP recognises 5’ss through U1 snRNA’s highly conserved 6bp sequence that base pairs to the 5’ss of the pre-mRNA
- U2 snRNP binds to the branch site in the intron by base pairing between a highly conserved sequence in U2 snRNA and the pre-mRNA. U2 snRNP binding requires hydrolysis
- A pre-assembled U4-U5-U6 tri snRNP joins the complex of U1, U2 and the mRNA precursor to form the spliceosome
- U6 disengages from U4 and undergoes an intramolecular rearrangement that permits base pairing with U2 as well as interaction with the 5’ end of the intron, displacing U1 from the spliceosome
- U6 disengages from U4 and undergoes an intramolecular rearrangement that permits base pairing with U2 as well as interaction with the 5’ end of the intron, displacing U1 from the spliceosome
- U5 aligns the free 5’ exon with the 3’ exon so that the 3’ hydroxyl group of the 5’ exon is positioned to nucleophilically attack the 3’ss to generate the spliced product
- U2, U5, and U6 bound to the excised lariat intron are released to complete the splicing reaction
6
Q
What is probably the catalytic centre of the spliceosome?
A
U2-U6 helix
