Mechanisms of Translation Flashcards
1
Q
Overview
A
- mRNA threads through subunit interface + is decoded
- 3 tRNA binding sites (A,P,E)
- Antibiotics
2
Q
Recognition of the tRNA + mechanism for correct amino acid
A
- Apart from tRNA initiator, all aminoacyl tRNAs interact w/ the ribosome + elongation factors in the same way
- 2o structure (RNA PolIII, 5’ end removed by RNase P, CCA added to 3’, short intron removed, bases modified)
- 3o structure (Non Watson-crick bp, triple base interaction, type I and II)
3
Q
Charging tRNAs
A
- Aminoacyl tRNA synthetase recognise both aa + tRNA
- Each tRNA has own E
4
Q
Mis-charging
A
- Mutating genes for tRNA → aa linked to wrong tRNA
- Mutations = in anticodon, D loop or acceptor stem of tRNA
- Freq of error rate = 10^-4 → 2 recognition steps
- 2 steps as recognition of aa by synthetase involves non-covalent interaction btw aa + E but some only differ by 1 methyl
- 2 steps separated by irreversible step to make frequency multiplicative
5
Q
Example - isoleucine tRNA synthetase
A
- Isoleucine binding in 1st step: binding of Ile + forming Ale-adenylate 10^2 x better than Val binding
- Rate of hydrolysis in 2nd: valyl-adenylate hydrolysed by E ↑ faster than isoleuc-adenylate, x proceed to tRNA
6
Q
Comparison of 30S + 50S subunits
A
- RNA domain fits together that determines 50S
- In AS + 30S interaction face = no proteins, ribozyme
- +vely charged tails on 12 proteins make interaction w/ RNA, stab
- 50S = monolithic
7
Q
Experimental evidence
A
- Free cell system, buffer and varying conc of soluble extract
- Yields ribosome carrying polyPhe-tRNA, ribosomes washed
- Incubated w/ puromycin
- 50% of ribosome release 14C labelled polyPhe-PM → ribosome itself forms peptide bond, x need E-supplying material
- Then was ribosome, add GTP, remove PM
- Remaining 50% of ribosome release 14C-Phe transferred → ribosomes have PM-reactive site + PM unreactive site
8
Q
Association of soluble factors to aa-tRNA
A
- Non-ribosomal = EFT/EFG
- EFT = EfTu (binds GTP) + EfTs (GEF, removes GDP from EfTu)
- EfTu = ↑ abundant, affinity is higher for GDP, only EfTu-GTP complexes w/ aatRNA
9
Q
Matching codon to anticodon
A
- Correct = 6 H bonds, incorrect = 5
- ‘wobble interaction’, even in correct orientation = imperfect
- G=U = acceptable at 3rd but not 1/2nd
- Wobbles take up ↑ space but little difference in binding E
10
Q
Different conformations of tRNA
A
- Ternary complex
- A*/T conformation (both cognate and non-cognate)
- A/T (only cognate, 30o bend in tRNA)
11
Q
How does the ribosome maintain accuracy?
A
- Mutations in ribosomal protein (S4,55+12 = close to decoding site)
- S4/5 = ram mutation, faster than WT
- S12 = Str mutation, slower than WT
- Ribosome has 2 conformations (open conf, E site is strong, A weak, alternative = opposite)
- Only cognate tRNAs cause closure of 30S
12
Q
Overall structure + maintaining accuracy
A
- Ribosomes decode mRNA codons by selecting aa-tRNAs delivered by Ef-Tu
- Both cognate + near cognate explore A of open 30S
- Initial codon-independent binding of ternary complex, codon recognition, GTPase activation
- G530 stabilises codon activation helix, step-wise latching of decoding centre, 30S closes, Ef-Tu docks w/ SRL, EfTu activated, accommodation of aa-tRNA
- Near-cognate x induce G530 latch, favours open 30S
- A minor interactions at pos I stabilise non-cognate W-C in enol tautomer
13
Q
Decoding
A
- Binding of aa complex to ribosome surface is weak, binding E of codon/anticodon critical for cognate vs non cognate
- Non cognate diffuse away
- G530, A1492/3 change position if P/A empty
- Interactions: A minor at pos I (A forms 4 H bonds w/ G-C, perfect geometry)
- A minor at 1 w/ wobble (A forms 2 H bonds w/ G-U, different geometry)
14
Q
Summary of decoding
A
- P/A kink, DC restricted allows geometry of nucl 1+ 2 of codon
- No restraint on 3
- G-U in pos 1 or 2 creates repulsion unless Enol tautomer
- Mutations can occur if make more space w/ mutations so accommodate wobble e.g. paromycin opens A site
15
Q
Domain closure
A
- 30S domain closure needs W-C bp at pos 1+2 anticodon
- G530 has 2 structures: semi-on (forms H bond w/ codon-anticodon) on (3A movement, H bond formed at A1492 at 2+3)
- Near cognate, G530 x stabilises codon-anticodon
- Sometimes G530 stabilises Watson crick like codon anticodon helix → miscoding
- Cognate: samples 30S → shoulder moves → Ef-Tu docks at SRL → GTPase active → G530 in semiON, → ON
- Near cognate: ternary complex samples A site of open 30S subunit w/o inducing 30S rearrangements