Kinetic proofreading
description
Transcript of Kinetic proofreading
Kinetic proofreadingJ.J. Hopfield 1974tRNA – 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
910errorP
(In human chromosome #1 there are ~200,000,000 base pairs )
Less than 1 error per strand
Affinities and ErrorsTypical hydrogen bond energy of codon-anticodon triplets ~ 5 kcal/mole
A U
UG
G C
molekcalGGG GUAU 1~
calTKB2110
18.010610
1000expexp 2321
TK
GPB
error
In order to get the observed error rates by energy difference alone:tRNA-mRNA:
DNA replication:
molekcalG 5.5
molekcalG 5.12
Michaelis – Menten Kinetics
SE ES 1k
1k
Enzyme Substrates Enzyme substrates complex
Product
EPSk 2
ESkkSEkdtESd
211
cPCccC Cw
k
k
c
c
'The desired enzymatic process
Hopfield’s problem
The undesired enzymatic process cPDccD D
wk
k
D
D
'
Assumptions:DC kk '' cDC - much smaller than
the other ratesw
0fekk
kwkw
PP RT
G
D
C
D
C
C
D
Steady state error rate is
embodied in the reaction rates
Hopfield’s Solution
Cwm
k
kPCcCccC
c
c
*''
cC
Cl
With these kinetics:
0fCcDc
Another option: one step and time dependent reaction rates.
And with: 0fll
D
C
negligible is 'w
kkm DC
20fP
P
C
D
Kinetic proofreading• Multistep process.• Discard step.• Directionality by energy expenditure.• Dominance of direct production.
CccCc
c
k
k
'
cPCc cw *
',mm
Cl 'Cl
cC
',mm
Proofreading - Protein Synthesis
GTP GDP+P
(Hopfield 1974)
• Fluorescently labeled tRNA molecules.• Antibiotic inhibitors of tRNA selection.• Nonhydrolizable GTP analogues.• Enzymatically and chemically altered ribosome complexes
Blanchard et al. 2004
Codon recognition state
GTPase activity stimulation (different rates, k3, for cognate and non-cognate) GTP hydrolysis Phosphate releaseProofreading
Experimental result – Protein synthesis
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
• Directionality through energy consumption
• Discard steps.• Multi-steps.
Specificity through energetic differences isn’t enough.
To achieve enzymatic proofreading:
Living cells need to regulate substance concentration and control reaction rates to achieve the conditions for the nest proofreading chain.