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Kinetic proofreading
tRNA – Ribosome analogy
J.J. Hopfield 1974
Outline
• High precision bio-synthetic processes
• The matching problem and its solution by
kinetic proofreading
• Examples and more recent results
tRNA-mRNA matching (protein synthesis)
Remember: coding redundancy
DNA replication
Perror  10
9
Less than 1 error per strand
(In human chromosome #1 there are
~200,000,000 base pairs )
Affinities and Errors
Typical hydrogen bond energy of codonanticodon triplets ~ 5 kcal/mole
A
U
In order to get the observed
error rates by energy
difference alone:
tRNA-mRNA:
G
C
kcal
G  5.5
mole
kcal
DNA replication: G  12.5
mole
kcal
G AU  GGU  G ~ 1
mole
K BT  1021 cal
G
U
Perror
 G 
1000


  exp   21
 exp  
 0.18
23 
 10  6 10 
 K BT 
Michaelis – Menten Kinetics
ES
Enzyme
 ES

k 1
Substrates
k1
Enzyme
substrates
complex
 PS  E
k2
Product
d ES 
 k1 E S   k 1  k 2 ES 
dt
Hopfield’s problem
The desired enzymatic process
The undesired enzymatic process
k 'c
C  c  Cc 
 PC
w
kc
k 'D
D  c  Dc 
 PD
w
kD
Assumptions:
C  D  c
Steady state error rate is
embodied in the reaction rates
k 'C  k 'D
 c 
 c 
w - much smaller than
the other rates
G


PD w  kC kC


 e RT  f 0
PC w  k D k D
Hopfield’s Solution
k 'c
C  c  Cc  Cc 
 PC
m'
*
w
kc
lC
C c
m'  k C  k D
w is negligible
With these kinetics:
Dc  
Cc
f0
lC
 f0 
And with:
lD
PD
2
  f0 
PC
Another option: one step and time dependent reaction rates.
Kinetic proofreading
•
•
•
•
Multistep process.
Discard step.
Directionality by energy expenditure.
Dominance of direct production.


k 'c
C  c  Cc
kc
Cc 
 Pc
*
m, m'
lC
l'C
C c
w
 c 
Proofreading - Protein Synthesis
GTP
(Hopfield 1974)
GDP+P
Experimental result – Protein synthesis
•
•
•
•
Blanchard et al. 2004
Fluorescently labeled tRNA molecules.
Antibiotic inhibitors of tRNA selection.
Nonhydrolizable GTP analogues.
Enzymatically and chemically altered ribosome complexes
GTPase activity stimulation
Codon recognition
(different rates, k3, for cognate
state
and non-cognate) GTP hydrolysis Phosphate releaseProofreading
Experimental result – tRNA & amino acid binding
Measuring concentrations in time of correct (isoleucine) and incorrect
(valine) charged tRNAs
Energy
expenditure
Correct /
incorrect?
DNA replication
Additional step forward function of the
enzyme (DNA polymerase)
Schaaper 1993
Conclusions and Key Points
Specificity through energetic differences isn’t enough.
To achieve enzymatic proofreading:
• Directionality through
energy consumption
• Discard steps.
• Multi-steps.
Living cells need to regulate substance
concentration and control reaction rates to achieve
the conditions for the nest proofreading chain.