What was the 1st experiment that indicated that RNA was synthesized as a longer precursor?
Conducted Pulse-Chase experiment: label RNA w/ 32P for 30 min & separate by rate-zonal centrifugation (thru sucrose gradient); stop transcription w/ actinomycin D (which stops Pol II) then separate by centrifugation; look @ size of RNA
What was the conclusion of the 1st experiment?
Size of RNAs was greatly reduced
What was the 2nd experiment that indicated that RNA was synthesized as a larger precursor?
Used sucrose gradient to look at total nuclear RNA & compare to mRNAs isolated from polyribosomes (all mRNAs that are being translated are much smaller in size)
What was the conclusion of the 2nd experiment?
mRNA is derived from hnRNA & this process results in an overall decrease in size of RNA (b/c of removal of introns)
Complex of RNA & proteins that is responsible for most pre-RNA splicing; composed of 5 snRNPs (U1,U2,U4/U6,U5)
Nucleotide sequences within the intron and at the borders between introns and exons which determine whether an exon is included in the mature mRNA; 5' splice site, 3' splice site, & branch point (which contains important A residue)
Small nuclear RNPs (snRNPs)
Small proteins, each w/ a single mRNA and binding site for 10+ proteins
What is the first step in splicing?
Recognition of the junction between exons & introns: RNA components in snRNPs recognize sequences @ splice sites by base-pairing
What snRNP binds to the 5' splice site?
U1-containing snRNP binds to GU sequence through base-pairing with U1 RNA along w/ U2AF (which binds to AG & flanking sequences @ 3' splice site)
What is the role of U2AF?
Binds to 3' splice site and recruits U2 snRNP to branch site & interaction between U1/U2 proteins brings together exons (2'OH of adenosine in branch point becomes nucleophile that attacks the phosphodiester bond @ 5' splice site)
What is the role of U4-U6-U5?
Trimeric snRNP binds, w/ U6 binding to U2 & catalyzes attack of branch point on the 5' splice site (A now has 3 phosphodiester bonds @ 5',3',2' sites, which forms lariat)
How is the intron released?
The intron is released as a lariat when the 3' OH attacks at 5' splice site on phosphodiester bond @ junction of 3' splice site
What are the 3 steps of RNA splicing?
1. Assembly of spliceosome
2. 2 Transesterification steps
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