Lecture 37: RNA Structure, Synthesis, Processing II Flashcards
Eukaryotic mRNA processing modifications
- 5’ cap
- 3’ polyA tail
- Intron splicing
mRNA processing locations
Eukaryotic mRNA has to be processed in the nucleus before export to cytoplasm, where translation occurs
Prokaryotic mRNA processing
Prokaryotes don’t do mRNA processing! Replication, transcription, translation are simultaneous and in the same place.
5’ cap
- GTP added backwards to 5’ end of mRNA
- Only 5’-5’ tri-Pi linkage
- Catalyzed by guanylyl transferase
5’ cap addition process
- γ Pi released from RNA
- PPi released from GTP
- Condensation
- N7 methylation in nucleus
5’ cap functions
- Protect vs nuclease digestion
- Scaffold for protein binding
- Efficient translation
3’ polyadenylation tail
- Protects 3’ end from degradation (stabilizes mRNA)
- Added during transcription termination
3’ polyA addition process
Eukary. transcription termination:
1. RNA poly puts AAUAAA cleavage signal to 3’ end
2. Specific endonuclease cleaves downstream
3. PolyA polymerase adds A’s w/ ATP
4. PolyA binding proteins associate
Intron splicing
- Uses small nuclear ribonuclear proteins (snRNPs)
- Uses unique 2’-5’ P-diester branch bond
Alternative splicing
Multiple proteins can be encoded in 1 gene; final expression depends on how transcript is spliced
snRNPs
Small nuclear ribonuclear proteins = snRNA + proteins (U1-U6)
- Ribozyme (RNA w/ enzymatic activity)
Intron structure
All introns start GU, end AG; consensus seq. at 3’ and 5’ exon/intron borders
Intron branch point
A located in pyrimidine rich seq., ~50 bases from intron 3’ end
snRNP roles
U1: binds 5’ splice site
U2: binds branch site (catalytic center)
U5: binds 3’ splice site, loops over to 5’ site
U4: temporary U6 catalytic center masking
U6: Catalyzes splicing
How does U1 bind the 5’ splice site?
U1 RNA is complementary to the 5’ splice site; many U1 families exist
Spliceosome assembly process
- U1 binds 5’ end, U2 binds branch site
- RNA folds, U4/5/6 complex added
- U1/U4 leave; U5 holds splice ends together
- U2/U6 exert catalytic activity
U2/U6 catalytic complex activity
U2/U6 ribozyme have complementary RNA to catalytic site
- Catalyze 2 transesterifications:
1. 2’ OH of A branch point attacks 5’ splice site
2. 3’ OH of exon 1 attacks 3’ splice site
Intron leaves as lariat (loop)
Beta thalassemia major, splicing pathology
Point mutations can create new splice sites (req. close enough to same branch point), introducing new stop codons e.g. no functional beta expression
Exon/Intron Splicing Enhancers vs Silencers
Enhancers bind SR proteins (Ser+Arg rich, favor splicing i.e. exon spliced in)
Silencers bind hnRNPs, inhib. splicing i.e. exon not spliced in
E.g. CD44 v5 exon 10 in cancer
tRNA
- Same in prokary/eukary.
- Features post-transcriptionally modified bases, not just AGCU
- Clover leaf structure w/ acceptor stem, anti-codon region
tRNA acceptor stem
Site of AA charging by synthetases
tRNA anti codon region
Interacts w/ mRNA
Bacterial tRNA features
- Multimeric (processed to monomer precursors)
- Cleaved 5’ and 3’ by RNAses
- Modified bases
- 3’ CCA added by tRNA nucleotidyl transferase
Prokaryote tRNA transcript processing
- 5’ end cleavage
- 3’ end cleavage
- Base modification
- CCA added to 3’ by nucleotidyl transferase
Eukaryotic tRNA features
- Monomers
- Req. removal of intron near anticodon
- Also 3’ CCA, base modifications
Prokaryotic ribosome subunits
50S + 30S = 70S
Eukaryotic ribosome subunits
60S + 40S = 80S
Ribosomal subunit composition
rRNA + proteins; medium rRNA forms smaller subunit
Prokaryotic rRNA to subunit
50S = 23S, 5S rRNA
30S = 16S rRNA
Eukaryotic rRNA to subunit
60S = 28S, 5.8S rRNA
40S = 18S rRNA
Prokaryotic rRNA synthesis
All rRNAs synth. 1 large precursor then cleaved
Eukaryotic rRNA synthesis
Poly I in nucleolus
Synth. as monomeric precursors from concatenated genes (moderately repetitive seq’s, many copies of gene-promoter joined together)
Creates 45S rRNA precursor which self-splices; subunits self-assemble in nucleolus -> separate until active in cytoplasm
Negative feedback of eukaryotic rRNA transcription
Excess 5S rRNA inhibits further transcription
