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Kinetic proofreading

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.

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Kinetic proofreading

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

  2. Outline • High precision bio-synthetic processes • The matching problem and its solution by kinetic proofreading • Examples and more recent results

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

  4. DNA replication Less than 1 error per strand (In human chromosome #1 there are ~200,000,000 base pairs )

  5. Affinities and Errors In order to get the observed error rates by energy difference alone: Typical hydrogen bond energy of codon-anticodon triplets ~ 5 kcal/mole tRNA-mRNA: A U DNA replication: G C G U

  6. Michaelis – Menten Kinetics Enzyme Substrates Enzymesubstratescomplex Product

  7. Steady state error rate is embodied in the reaction rates Hopfield’s problem The desired enzymatic process The undesired enzymatic process Assumptions: - much smaller than the other rates

  8. With these kinetics: And with: Hopfield’s Solution Another option: one step and time dependent reaction rates.

  9. Kinetic proofreading • Multistep process. • Discard step. • Directionality by energy expenditure. • Dominance of direct production.

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

  11. 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 GTPase activity stimulation (different rates, k3, for cognate and non-cognate) Codon recognition state GTP hydrolysis Phosphate release Proofreading

  12. Experimental result – tRNA & amino acid binding Measuring concentrations in time of correct (isoleucine) and incorrect (valine) charged tRNAs Energy expenditure Correct / incorrect?

  13. DNA replication Additional step forward function of the enzyme (DNA polymerase) Schaaper 1993

  14. 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.

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