Module 4 Section 2 Flashcards
1
Q
Processing of RNA Euk vs. Prok
A
- mRNA subject to constant degradation
- In prokaryotes, RNA translated before transcription is terminated
- In Euk, newly transcribed pre-mRNA subject to modifications prior to exit from nucleus
2
Q
If eukaryotic pre-mRNAs were not post-transcriptionally modified, what would be the consequence?
A
- enhanced degradation (end caps)
- Impaired ribosome binding
- Translation of non-coding DNA sequences
3
Q
Processing of pre-mRNA
A
- 5’ capping
- 3’ polyadenylation
- splicing
- nucleotide editing
4
Q
5’ Capping
A
- 7-methyl guanosine attached to mRNA through 5’-5’-triphosphate linkage
- mediated by guanylyltransferase
- additional methyl groups often added to 2’OH of ribose sugars at the 1st and 2nd nucleotides adjacent to cap, and to N7 on the guanine (the cap one)
- occurs early in transcription (after addition of 20-30 nucleotides)
- Mediated by C-term domain (CTD) of RPBI
- Phosphorylation of CTD recruits specific RNA modifying enzymes (including guanylyltransferase)
- Cap-binding complex (CBC) recruits mRNA to ribosome
5
Q
Purpose of 5’ cap
A
- prevents recognition of the 5’ end by exoribonucleases
- mediates binding of mRNA to the ribosome
6
Q
3’ polyadenylation steps
A
- Pol II transcribes the poly(A) addition site at term end of transcript
Marked by two sequence elements:
-highly conserved sequence 5’-AAUAAA-3’ 10-30 nucleotides upstream of cleavage site
-GT rich region 20-40 nucleotides downstream of cut site - Polyadenylation factors bind poly(A) signal upstream of cleavage site
-cleavage generates free 3’OH that defines the end of mRNA - “A” residues are added to free 3’OH immediately in Poly(A) polymerase (PAP) catalyzed rxn
-A-residues are bound by Poly(A) binding proteins (PABP) which protect 3’mRNA from exoribonucleases
7
Q
mRNA circularization
A
- 5’ cap and 3’ polyA tail associate with subunits of elF4F to circularize mRNA
1. mediates ribosome binding
2. promotes translational efficiency
3. ensures complete processing
8
Q
Splicing of Pre-mRNA
A
-exons: typically <1000 nucleotides Introns: -50-20000 nucleotides -do not code for protein -contain regulatory sequences -allow for alternative splicing of genes 5'UTR and 3'UTR are NOT spliced out
9
Q
Splicing
A
- the controlled act of breaking and joining specific phosphodiester bonds to excise introns
- cannot reorder RNA (ie. cannot have 2, 1, 3)
10
Q
Spliceosome
A
- mediates splicing
- complex of 5 small nuclear ribonuclear proteins (snRNPs)
- snRNP = snRNA and protein subunits
11
Q
Reverse transcription
A
- generation of cDNA
- make use of 3’polyA to make sure only mRNA is replicated
- use polyT sequence as a primer (Oligo dT primer)