Kirill B. Gromadski and Marina V. Rodnina

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Kinetic Determinants of High-Fidelity
Discrimination on the Ribosome

• Kirill B. Gromadski and Marina V. Rodnina
• Biochemistry 4000
• Dora Capatos

2
tRNA Selection
50S subunit
30S subunit
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
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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

4
tRNA Discrimination on the Ribosome

• Rejection of incorrect tRNAs occurs in 2 stages
• 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 pairs 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 ribosome

Mg2 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 fragments
• Of 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 ?-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
• Syringe ribosomes in excess
• Syringe Ternary complex
• tRNA-labelled (fluorescence or radioactive
  isotope)
• 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 ltlt ribosome to
  ensure that only one round of selection occurs

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Initial Binding
Kapp Increases linearly with Ribosome

• 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

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Codon Recognition
Near-cognate
Cognate

• K2 190 20 s-1

• Kapp determined from fluorescence increased with
  ribosome concentration in a hyperbolic shape
• Kapp increased faster for cognate vs.
  near-cognate tRNAs

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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

• Initial binding of ternary
• complex reversible when
• there is no match between
• codon and anticodon
• Cognate dissociates very
• slowly compared to
• near-cognate

1 k-2 0.23 0.05 s-1 (Cognate) ? k-2
0 2 k-2 80 15 s-1 (Near-cognate) 3
Control no dissociation occurs upon addition of
buffer instead of non-fluorescent Ternary
complex
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GTPase Activation GTP Hydrolysis
Saturates at 110 25 s-1

• 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

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GTPase Activation GTP Hydrolysis
Kapp 62 /- 3 s-1 (UUU codon) Kapp 0.35 /-
0.02 s-1 (CUC)
absence of ribosomes
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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 ternary complexes, 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/mol GTPase
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 gt 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/Km For
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.