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Kinetic proofreading

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J.J. Hopfield 1974. tRNA – Ribosome analogy. Kinetic proofreading. 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. - PowerPoint PPT Presentation

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  • 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.