Kinetic Determinants of
High-Fidelity Discrimination on
the Ribosome
Kirill B. Gromadski and Marina V. Rodnina
Biochemistry 4000
Dora Capatos
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
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
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
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
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
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
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.
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!
Objective
What are the respective contributions of
initial selection and proofreading to tRNA
selection that account for the low error
rate of the ribosome?
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
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)
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.
Kinetic Mechanism of
EF-Tu-Dependent aa-tRNA Binding
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.
Individual Steps of Selection
• Monitor GTP hydrolysis & peptide bond
formation by quench flow using isotopes [γ32GTP or aa-tRNA charged with 3H- or 14Clabelled 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
Experimental Setup
•
1.
2.
3.
Measure binding or dissociation:
Syringe: ribosomes in excess
Syringe: Ternary complex
tRNA-labelled (fluorescence or radioactive
isotope)
4. Use high fidelity buffer conditions (low Mg 2+
concentrations)
5. Do stopped flow or quench flow experiments
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
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
Codon Recognition
Near-cognate
Cognate
• 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
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
Dissociation of Codon-Recognition
Complex
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 nonfluorescent Ternary complex
•Initial binding of ternary
complex reversible when
there is no match between
codon and anticodon
•Cognate dissociates very
slowly compared to
near-cognate
GTPase Activation & GTP Hydrolysis
Saturates at 110 ± 25 s-1
•For cognate tRNA, Kapp increased with
ribosome concentration
•For near-cognate, kapp was constant at
0.4 ± 0.1 s-1 throughout the titration
•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
GTPase Activation &
GTP Hydrolysis
= absence of ribosomes
Kapp = 62 +/- 3 s-1 (UUU codon)
Kapp = 0.35 +/- 0.02 s-1 (CUC)
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)
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
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)
Cognate vs. Near Cognate Binding
Efficiency of initial selection = Kcat/Km
For cognate tRNA, Kcat = K2
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
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. nearcognate substrate
• Thermodynamic stability differences between
cognate and near-cognate tRNAs: RTln350 is
the ratio of rate constants: k-2near cognate /k2cognate and 650 for GTP hydrolysis gives
RTln(650) = 2.7 kcal/mol.
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
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
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
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.
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
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.