Kinetic Determinants of High-Fidelity Discrimination on the Ribosome
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Kirill B. Gromadski and Marina V. Rodnina
Biochemistry 4000 Dora Capatos
Kinetic Determinants of High-Fidelity Discrimination on
the Ribosome
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tRNA Selection
Ribosome selects aminoacyl transfer RNA (aa-tRNA) with anticodon matching to the mRNA codon in the A site from the bulk of nonmatching aa-tRNAs
30S subunit
50S subunit
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Mismatches• Cognate tRNA: matches codon in the
decoding site• Near-cognate tRNA: one mismatched
base pair• Frequency of mismatch is 10-3 to 10-4
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tRNA Discrimination on the Ribosome
• Rejection of incorrect tRNAs occurs in 2 stages:
1. Initial selection of ternary complexes EF-Tu-GTP-aa-tRNA
2. Proofreading of aa-tRNA
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What is Initial Selection? • Steps of codon recognition and GTPase activation• Codon recognition occurs when the first codon-anticodon
base pair is stabilized by binding of the rRNA A1493 base pair’s minor groove in the decoding centre
• These interactions enable the ribosome to monitor whether an incoming tRNA is cognate to the codon in the A site.
• A non Watson-Crick base pair could not bind these ribosomal bases in the same way.
• An incorrect codon-anticodon provides insufficient free energy to bind the tRNA to the ribosome and it dissociates from it, still in its ternary complex with EF-Tu and GTP
• Occurs prior to GTP hydrolysis and must be fast
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GTPase Activation & Hydrolysis• GTPase activation of EF-Tu• Release of inorganic phosphate induces conformational
transition of EF-Tu from GTP to GDP form• EF-Tu in GDP form loses affinity for aa-tRNA and
dissociates from the ribosomeMg2+ ion
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Accommodation
• After GTP hydrolysis, EF-Tu loses its affinity for aa-tRNA and the aminoacyl end of aatRNA is free to move into the peptidyl transferase centre on the 50S subunit
• tRNA accommodation occurs in the A site• Occurs when EF-Tu hydrolyzes its bound GTP
to GDP + Pi and is released from the ribosome permitting the aa-tRNA to fully bind to the A site
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Proofreading• Proofreading step is independent of the initial
selection step• Proofreading includes the conformational
changes that occur after GTP hydrolysis and before peptide bond formation
• Rejection will occur if a mismatch is detected, and the aa-tRNA will dissociate from the ribosome
• Otherwise, peptide bond formation will occur.
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The Decoding Problem
Crystal structure of 30S subunit with anticodon stem-loop fragmentsOf tRNA bound to codon triplets in the decoding site show that the codon-anticodon complex forms interactions with rRNA in the decoding site.
Free energy of Watson Crick base pairing alone cannot account for the high efficiency of tRNA selection!
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Objective
What are the respective contributions of initial selection and proofreading to tRNA
selection that account for the low error rate of the ribosome?
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I. Overall Selectivity• Measure selectivity of the ribosome at high
& low fidelity conditions:– Conditions at which overall fidelity of selection was
high due to high efficiency of both initial selection and proofreading
– Overall selectivity measured by competition between Leu-tRNAleu specific for the CUC codon
– Measure proofreading by
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Results: Selectivity of the Ribosome
Since initial selection and proofreading steps are independent:
Probability of Overall Selection = Prob (Initial Selection) x Prob (proofreading)
At high fidelity: 1/450 = (1/30 x 1/15)
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Results: Error Rates?• Contribution of initial selection is calculated from
overall selectivity to be about 30. Proofreading was calculated to be about 15.
• Overall selectivity is product of initial selection and proofreading and
• is approximately 450 at high fidelity conditions.
• Incorporation of 1 incorrect per 450 amino acids• This indicates an efficiency of initial selection of 30.
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Kinetic Mechanism of EF-Tu-Dependent aa-tRNA Binding
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II. Individual Steps of Selection
• Elemental rate constants of the steps contributing to initial selection of ternary complex EF-Tu-Phe-tRNAPhe
(anticodon 3’-AAG-5’) were determined on mRNA programmed (initiated) ribosomes with cognate (UUU) or near-cognate (CUC) codons in the A site.
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Individual Steps of Selection• Monitor GTP hydrolysis & peptide bond
formation by quench flow using isotopes [γ-32GTP or aa-tRNA charged with 3H- or 14C-labelled amino acids
• All other rate constants measured by fluorescence experiments carried out by stopped-flow technique (measure conformational changes)
• Fluorophores are wybutine (binds to tRNA) and proflavin
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Experimental Setup
• Measure binding or dissociation:1. Syringe: ribosomes in excess2. Syringe: Ternary complex 3. tRNA-labelled (fluorescence or radioactive
isotope)4. Use high fidelity buffer conditions (low Mg 2+
concentrations)
5. Do stopped flow or quench flow experiments
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Rapid Kinetics
• Apparent rate constants• Do not follow Michaelis Menten Kinetics; must
use mathematical curve fitting to obtain kapparent
• Pre-steady state conditions• Use stopped flow or quench flow device • Single turnover conditions: [TC] << [ribosome] to
ensure that only one round of selection occurs
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Initial Binding
•R + TC Complex
•k1 is 2nd Order•K-1 is 1st Order•K1 = 140 +/-20 uM-1 s-1 (slope)
•KM = (k2 + k-1) / k1•KM ~ [ribosome] at ½ Vmax
•Exponential curve Fitting
Kapp Increases linearly with [Ribosome]
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Codon Recognition
• Kapp determined from fluorescence increased with ribosome concentration in a hyperbolic shape
• Kapp increased faster for cognate vs. near-cognate tRNAs
•K2 = 190 ± 20 s-1
Near-cognate
Cognate
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Chase Experiments• To a fluorescently labelled Phe-tRNA in
complex with GTP and GTPase deficient EF-Tu(H84A), initiate dissociation by adding an excess of nonfluorescent ternary complex and monitor fluorescence decrease over time
• Use GTPase deficient EF-Tu to determine if GTP hydrolysis has an effect on fluorescence
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Dissociation of Codon-RecognitionComplex
1 = k-2 = 0.23 ± 0.05 s-1 (Cognate) k-2 ~ 02 = k-2 = 80 ± 15 s-1 (Near-cognate)3 = Control: no dissociation occurs upon addition of buffer instead of non-fluorescent Ternary complex
•Initial binding of ternary complex reversible when there is no match betweencodon and anticodon
•Cognate dissociates very slowly compared to near-cognate
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GTPase Activation & GTP Hydrolysis
•Measured using fluorescent GTP derivative, mant-GTP
•Kapp measured by GTP hydrolysis represent rate k3 for GTPase activation assuming no rate limiting step preceding GTPase activation
•For cognate tRNA, Kapp increased with ribosome concentration
•For near-cognate, kapp was constant at 0.4 ± 0.1 s-1 throughout the titration
Saturates at 110 ± 25 s-1
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GTPase Activation & GTP Hydrolysis
= absence of ribosomesKapp = 62 +/- 3 s-1 (UUU codon)Kapp = 0.35 +/- 0.02 s-1 (CUC)
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Proofreading & Peptide Bond Formation
Kapp = 6.6 +/- 0.4 s-1 (Cognate)Kapp = 0.19 +/- 0.04 s-1 (Near cognate)
Proofreading = fraction of dipeptides that undergo peptidyl transfer = k5 /(k5 + k7)
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Kinetic Determinants of Initial Selection
k1, k-1, k2, were for the same for cognate and near-cognate ternarycomplexes, thus the only rate constant that contributes to the different affinity is k-2. So k-2 near cognate /k-2cognate = 80/0.23 ≈ 350.Free energy difference: ∆∆Go = -RTlnk = -RTln(350) = 3.4 kcal/molGTPase activation of EF-Tu is rate limiting for GTP hydrolysis
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Kinetic Determinants of Initial Selection
•GTP hydrolysis by EF-Tu regulates initial selection•K3cognate/k3near-cognate = 650 => 650-fold GTP hydrolysis of cognate compared to near cognate •K1 and K2 do not reach equilibruim (would be too slow otherwise)
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Cognate vs. Near Cognate Binding
Efficiency of initial selection = Kcat/KmFor cognate tRNA, Kcat = K2
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Summary• Both initial selection prior to and proofreading
after GTP hydrolysis are required for efficient tRNA discrimination in vitro.
• Fidelity of initial selection:Finitial selection = 60 ± 20 is close to 30 • Rate constants of GTPase activation and tRNA
accommodation in the A site are much faster for the correct than the incorrect substrates
• k1, k-1, k2, were for the same for cognate and near-cognate ternary complexes
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Discussion
• Thermodynamic vs. Kinetic Discrimination?
• tRNA selection at the initial selection step is kinetically controlled and is due to much faster (650-fold) GTP hydrolysis of cognate vs. near-cognate substrate
• Thermodynamic stability differences between cognate and near-cognate tRNAs: RTln350 is the ratio of rate constants: k-2near cognate /k-2cognate and 650 for GTP hydrolysis gives RTln(650) = 2.7 kcal/mol.
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Discussion• An incorrect codon-anticodon provides
insufficient free energy to bind the tRNA to the ribosome and it therefore dissociates from it, still in its ternary complex with EF-Tu and GTP bound
• Free energy of base-pairing alone is insufficient to discriminate between cognate (correct) and near-cognate (incorrect) tRNAs
• May differ by as little as a single mismatch in the codon-anticodon duplex
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Discussion• GTPase activation of EF-Tu requires precise
alignment of catalytic groups in active sites
• Changes of ribosome structure caused by the correct substrate may not occur or may be different with an incorrect substrate
• Reflect finding that rate constants of GTPase activation and tRNA accomodation in A site are much faster for correct vs. incorrect substrates
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Discussion• A-site binding is a non-equilibrium process that
is driven by the rapid irreversible forward reactions of GTP hydrolysis and peptide bond formation
• Discrimination is based on the large differences in the forward reaction rates of GTPase activation and accomodation
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Discussion• Induced Fit Model • Ribosome may be capable of preferential
stabilization of complexes with the correct substrate in both ground state and transition state
• Incorrect substrates may be poorly or not at all stabilized
• Suggests ribosome increases selection potential by checking structure of intermediates by an induced fit mechanism.
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Future Questions• Further structural studies
-Solve structure of the codon-anticodon complex in the decoding centre at high resolution
• Investigate induced fit discrimination mechanism of the ribosome
• Structure of conformational changes in proofreading
• Structural determinants that sense cognate base pairing
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References• Gromadski, K.B., Rodnina, M.V. 2004. Mol. Cell 13: 191-200.• Rodnina, M.V., Gromadski, K.B., Kothe, U., Wieden, H. FEBS Lett.
579: 938-942.• Rodnina, M.V., Wintermeyer, W. 2001. TIBS 26 (2): 124-130.• Voet, D., Voet J. 2004. Biochemistry. Wiley, New York.