Kinetic proofreading

Kinetic proofreadingJ.J. Hopfield 1974tRNA Ribosome analogy

OutlineHigh precision bio-synthetic processes The matching problem and its solution by kinetic proofreadingExamples and more recent results

tRNA-mRNA matching (protein synthesis)Remember: coding redundancy

DNA replication

Affinities and Errors

Michaelis Menten KineticsEnzymeSubstratesEnzyme substrates complexProduct

Hopfields problem

Hopfields SolutionAnother option: one step and time dependent reaction rates.

Kinetic proofreadingMultistep process.Discard step.Directionality by energy expenditure.Dominance of direct production.

Proofreading - Protein Synthesis(Hopfield 1974)

Fluorescently labeled tRNA molecules.Antibiotic inhibitors of tRNA selection.Nonhydrolizable GTP analogues.Enzymatically and chemically altered ribosome complexesCodon recognition stateGTPase activity stimulation (different rates, k3, for cognate and non-cognate)GTP hydrolysisPhosphate releaseProofreadingExperimental result Protein synthesis

Experimental result tRNA & amino acid bindingMeasuring concentrations in time of correct (isoleucine) and incorrect (valine) charged tRNAsEnergy expenditureCorrect / incorrect?

DNA replicationAdditional step forward function of the enzyme (DNA polymerase)

Conclusions and Key PointsDirectionality through energy consumptionDiscard steps.Multi-steps. Specificity through energetic differences isnt 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.

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