Kinetic proofreadingJ.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

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

mole

kcalGGG GUAU 1~

calTKB2110

18.010610

1000expexp

2321

TK

GP

Berror

In order to get the observed error rates by energy difference alone:tRNA-mRNA:

DNA replication:

mole

kcalG 5.5

mole

kcalG 5.12

Michaelis – Menten Kinetics

SE ES 1k

1k

Enzyme Substrates Enzyme substrates complex

Product

EPSk 2

ESkkSEkdt

ESd211

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

0fek

k

kw

kw

P

P 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:

0fCc

Dc

Another option: one step and time dependent reaction rates.

And with: 0fl

l

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.