RNA transcription Flashcards
Where does the capping protein start and then where does it go?
Starts on RNA pol tail, then caps 5’ end of RNA and stays there until translation.
Processing factors
e.g. spliceosome, etc. Recruited by pattern of phosphorylation on c-term RNA pol tail
What do splicing proteins do?
remove introns, join exons
What do 3’ end processing proteins do? What are some of their other effects?
Recognize cleavage/polyadenylation signals in mRNA, cleave at 3’ end. Destabilizes RNA pol
How does RNA pol dissociate?
3’ end processing proteins destabilize, then it’s dephosphrylated by phosphotases
Benefits of RNA splicing
Exon shuffling from recombination can generate novel domains, more coding potential with alternative splicing
Alternative splicing
Some exons are spliced out in between introns sometimes
Consensus sequences in pre-mRNA
GU on 5’ end of intron, ‘A’ at branch site, AG at 3’ end
Lariat formation
Two transesterifications
No energy req for transesterification
New bond on 2’ deoxyribose of ‘A’ branch site from G in GU
snRNP’s stay on it when it leaves
Mechanism of splicing
- U1 snRNP to 5’ splice site, U2AF/BBP to branch site
- U2 snRNP replaces U2AF/BBP
- U4/U6 snRNP come in with U5, form the loop
- U1 replaced by U6, branch site with U2 comes in at 5’ splice site
- U6 at 5’ splice site goes to 3’ splice site and breaks the loop off
What are spliceosomes?
ribonucleoprotein complexes. Contain snRNA’s that base pair.
Where does the Exon Junction Complex bind?
Binds to mRNA at the place where the intron used to be and stays there
RNA-RNA rearrangements in spliceosome - what rearranges and what’s needed to do it?
When U1 is replaced by U6, ATP is needed.
What are snRNA’s/snRNP’s?
Small nuclear RNA, U1-U6. Complexed with protein subunits to form snRNP’s.
Exon-marking proteins
Sit on exons while mRNA is being transcribed
Poly-A Binding Proteins
Sit at 3’ end, help with poly-A tail
What do 3’ end processing proteins do and when? Where are they before that?
Sit at RNA pol tail, then cleave at 3’ end once polyadenylation/cleavage signals happen
What happens when RNA pol dissociates?
Dephosphorylated by phosphotases, then can go do more transcription
polycistronic
Encodes more than one gene
What is different about prokaryotic mRNA compared to eukaryotic?
polycistronic, no exons, small noncoding sequences in the middle, no cap or AAA tail
Eukaryotic mRNA (compared to prok)
5’ cap > 5’ untranslated region > coding sequence > 3’ UTR > poly-A tail
UTR
Untranslated region in eukaryotic mRNA. This is how ribosomes recognize it
5’ cap structure
5’-5’ triphosphate bridge, then 7-methylguanosine attached to its 2’ carbon. Bound by cap-binding complex.
Cleavage factor
protein factors that cleave mRNA at the 3’ end when it’s done transcribing
Polyadenylation factor
Help add poly-A tail
Cleavage/polyadenylation factor similarities
Proteins that bind to signals coded in DNA, bind to RNA sequences as soon as RNA pol makes them.
Poly-A polymerase
Jumps on and makes poly-A tail when polyadenylation factors bind to RNA
Poly-A binding protein
Binds to poly-A tail to show that it’s ready to export. Lots of them bind, it’s not just one across the whole thing
Where do cleavage/polyadenylation factors start?
RNA pol c-terminus tail
mRNA as it goes through the nucleus
Still has exon-marking proteins, exon junction complex, a nuclear export receptor, poly-A binding proteins, cap binding complex and 5’ cap.
Some proteins leave as it goes through
mRNA right after it exits nuclear pore
Initiation factors for protein synth bind to it (elF4G, elF4E), then nonsense-mediated decay, then translation. Nuclear-specific proteins leave.
When does pol tail phosphorylation happen and why is it important?
Happens gradually during transcription initiation. Allows new proteins to bind to the tail.
What distinguishes mRNA from other RNA molecules?
5’ cap
What letter sequence is at the 3’ splice site?
AG (GU at 5’)
How do snRNP’s connect to intron splice sites?
base-pairing
What’s the first phosphoryl-transferase reaction in splicing?
U6 displaces U1, letting active site catalyze it. Doesn’t need ATP
What fidelity mechanisms increase splicing accuracy?
- Splicing happens during transcription (snRNP’s only have one site to bind to at a time)
- Exon definition: SR (‘serine/arginine’) proteins bind to
Why are SR proteins called that?
They have lots of serine / arginine
What do hnRNP proteins do?
Bind to introns to signal mRNA hasn’t been fully processed
What do SR proteins do?
Attach to newly made mRNA to stop it from binding to DNA strand
