Kinetic proofreading
J.J. Hopfield 1974 tRNA – Ribosome analogy

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
P error

10

9
Less than 1 error per strand
(In human chromosome #1 there are
~200,000,000 base pairs )

G
A
G
Affinities and Errors
Typical hydrogen bond energy of codonanticodon triplets ~ 5 kcal/mole
U
C
In order to get the observed error rates by energy difference alone: tRNA-mRNA:

G

5 .
5 kcal mole
DNA replication:
G
AU

G
GU

G


12

G ~
.
5 kcal
1 mole
K
B
T

10

21 cal
U
P error
 exp
 

K
G
B
T


 exp



1000
10

21 
6

10
23



0 .
18

Michaelis – Menten Kinetics
E

S
Enzyme Substrates

 k

1
ES
Enzyme d dt

 k
2 P
S
Product
 substrates k
1 complex   

 k

1
 k
2
  
E

Hopfield’s problem
The desired enzymatic process
The undesired enzymatic process
C
 c k k
'
 c c
Cc
 w
D
 c k k
'
D 
D
Dc
 w
P
C
 
P
D
 
Assumptions: k '
C
 k '
D w - much smaller than the other rates
Steady state error rate is embodied in the reaction rates

D

C
 w
 k
C w
 k
D
 k
C k
D
 e


G
RT
 f
0

Hopfield’s Solution
C
 c k
 k c
' c
Cc
 m '
Cc
*  w
P
C l
C m '
 k
C
 k
D
C
 c w is negligible
With these kinetics:
 
 
 f
0
And with: l
C l
D
 f
0


D
P

C

 
0
2
Another option: one step and time dependent reaction rates.

Kinetic proofreading
• Multistep process.
• Discard step.
• Directionality by energy expenditure.
• Dominance of direct production.
C
 c k
 k c
' c
Cc m , , m ' '
Cc
*  w l
C l '
C
C
 c
P c
 

Proofreading - Protein Synthesis
GTP GDP+P
(Hopfield 1974)

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 state
GTPase activity stimulation 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.