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Paper 3 > Mechanisms of Translation > Flashcards

Mechanisms of Translation Flashcards

1
Q

Overview

A
  • mRNA threads through subunit interface + is decoded
  • 3 tRNA binding sites (A,P,E)
  • Antibiotics
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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)
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3
Q

Charging tRNAs

A
  • Aminoacyl tRNA synthetase recognise both aa + tRNA

- Each tRNA has own E

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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
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5
Q

Example - isoleucine tRNA synthetase

A
  1. Isoleucine binding in 1st step: binding of Ile + forming Ale-adenylate 10^2 x better than Val binding
  2. Rate of hydrolysis in 2nd: valyl-adenylate hydrolysed by E ↑ faster than isoleuc-adenylate, x proceed to tRNA
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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
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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
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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
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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
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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)
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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
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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
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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)
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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
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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
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16
Q

Selection and accommodation

A
  • 30S works w/ 50S to ensure accommodation of cognate aa-tRNA in P + move tRNA
  • Using FRET, work constant of 2 steps
  • Shoulder lifted + stabilised on binding cognate tRNA during selection
  • Fastening cognate tRNA A site → additional intersubunit bridge
  • L25 holds tRNA in A site (x near-cognate)
  • ## Near-cognate produces distorted protein extensions in A site
17
Q

Proofreading

A
  • Entire anticodon loop monitored by nuc. from 16S rRNA + 23S rRNA
  • Elbow region scanned by rRNA + ribosomal protein determines whether to accom. acceptor tRNA
18
Q

Decoding centre activates Ef-Tu accomodation

A
  • G530 shifts to ‘latch’ to decoding centre → 30S closure
  • Accom = movement of aa-tRNA after Ef-Tu release into peptidyl transferase centre, CCA moves 100A
  • Puromycin stab decoding centre
  • 23S RNA active-site residues adopt different conformation depending on A site occupancy (w/o substrate, ribosome protects P site)
19
Q

Peptidyl transferase reaction

A
  • Need Eftu.GDP to be dissociated from the ribosome (x need E)
  • Catalytic centre of ribosome = just RNA, enhances peptide
  • 4x10^6 rate enhancement
  • Proton-shuttling via P-site adenosine 2’OH of A76
  • Antibiotics
20
Q

Translocation

A
  • Due to peptide bond leaving deactyl tRNA in P site + lengthened tRNA in A
  • EFG needed, binds A site
  • EFG mimics ternary complex
  • EFG departure leaves bs preformed for acceptance of ternary complex
  • peptidyl tRNA moves A→P, deaceylated tRNA moves p→E
  • New codon in A site
  • Ribosome can bind another EfTu.GDP but x another EfTu
  • Need to break H bonds btw tRNA-mRNA in A site
  • EFG catalyses tRNA translocation by disrupting interactions btw decoding centre + codon-anticodon duplex
21
Q

Exit tunnel

A
  • Stretches from peptidyl transferase + ends back of large subunit
  • SRP interacts w/ protein at end of tunnel
  • Around 100A long, accommodates diameter of a helix
  • Co-translational proteins bind
  • Antibiotics
22
Q

Initiation

A
  • 70S initiates
    1. Vacant 30S binds IF3 + 2, IF1 blocks A site, A1492/3 buried in/btw IF1/2
    2. fMet tRNA recruited, binds close to IF2
    3. mRNA binds, need correct Aug for initiator, 2o affects rate
    5. 2o unfolded facilitated by IF2-GTP
    6. Isomerisation of 30S allows P codon-anticodon interaction
    7. 30S docked by 50S
    8. GTPase of IF2 activated, GTP hydrolysed
    9. IF3 + 1 dissociate, IF2 undergoes conf change
    10. FMet-tRNA adjusted on ribosome occupying P/1
    11. Pi dissociates from IF2-GDP, helix-exit transition in switch II
    12. IF2 leaves
    13. Ef-Tu-GTP complex binds 70S, delivers ribosomal A site aminoacyl tRNA
  • Fmet tRNA bound in P site of 50S donates formyl-meth to A site bound aminoacyl - tRNA
23
Q

Termination of translation

A
  • Peptide elongated as peptidyl tRNA but cells have no peptidyl tRNA, released by hydrolysis
  • Chain terminating codons in A site
  • Protein factors release NfMet: RF1 recognises UAG/UAA, RF2 recognises UAA + UGA
  • RF1/2 bind A site, peptide released via hydrolytic cleavage
  • RF3 binds RF1/2 w/ GDP, GTP displaces GDP, RF3 released
  • Release factors alter specificity of peptidyl transferase (in elongation, peptidyl transferase transfers peptide from ester linkage w/ tRNA to peptide w/ incoming NH2)
  • Release factors have conserved GGQ, H bond distance from A76
  • Upon recognition of stop, conserved Q in GGQ inserted into catalytic centre
24
Q

RF1-3

A
  • RF1/2 stay on ribosome, need to be recycled
  • RF3 froms complex w/ GDP or GTP
  • RF3.GDP binds ribosome/RF1/2 complex weakly, GTP means binds tightly + displaces RF1/2
  • Hydrolysis of GTP liberates RF3/GDP, ribosome release factor releases deacylated tRNA
  • Efficiency of termination depends on nucleotide to the 3’ side of termination codon
25
Q

Rates + antibiotics

A
  • Rate = important for accuracy
  • Similarity btw eukaryotes + prokaryotes (all go into A site, long projection, molecular mimic)
  • Puronycin = chain termination
  • Fusidic acid = translocation
26
Q

Extra - summary

A
  • Initiation needs 30S, 50S, IF1-3
  • 30S carrying initiation factors binds initiation site → initiation complex
  • IF-3 released so 50S joins 30S mRNA complex
  • tRNA of 30S bacterial ribosomal subunit has a complementary sequence that bp SD during initiation
  • NH2 of initiator tRNA has formulated methionine
27
Q

Extra - Fmet + IF2

A
  • In initiation complex, small subunit alone is bound mRNA
  • Initiation codon lies within P site carried by small subunit
  • Large subunit binds, tRNA bs converted to P/A sites
  • Initiation = when AUG/GUG lies within ribosome binding site, only initiator tRNA enters partial P site
  • Accessory factors are critical
  • All aminoacyl tRNAs associate w/ ribosome by binding to an accessory factor
  • Ef-Tu places amino-acyl tRNA in A site, x bind fmet-tRNA
28
Q

Extra - Initiation in eukaryotic mRNA

A
  • Small subunit 1st recognises 5’ end of mRNA, moves to initiation site where joined by larger
  • 1st feature recognised = methylated 5’ cap
  • 40S recognises 5’ then migrates along mRNA
  • Melts 2o structure
  • Migration stops when 40S encounters AUG start in right context
29
Q

Extra - elongation factors

A
  • e1F2 + met-RNAi, e1F1,3 +A bind 40S → 43S preiniaiton complex
  • e1F4A,B,E,G bind 3’ of mRNA → capping binding complex
  • This complex associates w/ 3’ end of mRNA via e1FG
  • 43S binds initiation factors at 5’ end of mRNA + scans for initiation codon
  • 60S x work until E1F2/3 are released, facilitated by EIF5B
  • Once ribosome assembled, aa-tRNA enters A site w/ help of Ef-Tu
  • Dissoc once amino acyl tRNA in place
  • Ef-Tu-GTP binds aa-tRNA → ternary complex
  • Anticodon binds A site of 30S, codon-anticodon recognition triggers conf Δ
  • Ef-Tu releases amino acyl site, amino acyl ends twins closed
  • Peptide bond synthesis makes deacylated tRNA In P site + peptidyl tRNA in A
  • Translocation
30
Q

Extra- elongation factors bind alternatively to ribosome

A
  • Ef-Tu + Ef-G x bind at the same time
  • Ef-Tu released before Ef-G binds, then Ef-G released before aa-tRNA-Ef-tu-GTP binds
  • Structure of Ef-G mimics Ef-Tu bound to acceptor stem, compete for same bs?
  • Re-orientation of Ef-G occurs at translocation
  • Head of 30S swivels around neck when complete ribosome forms

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