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Kinetic proofreading

This outline explores the concept of kinetic proofreading as a mechanism for high-precision bio-synthetic processes. It delves into the matching problem in tRNA-mRNA interactions, emphasizing the importance of error correction. The text discusses the energy considerations in coding redundancy, DNA replication, and protein synthesis to maintain low error rates. Michaelis-Menten kinetics and the principles of enzyme-substrate interactions are examined in the context of Hopfield's problem, highlighting the desired enzymatic processes and the role of kinetic proofreading. Experimental results in protein synthesis and DNA replication reinforce the significance of energy expenditure and multi-step processes for accurate enzymatic proofreading. The conclusion underscores the necessity for living cells to regulate substance concentrations and control reaction rates for efficient proofreading mechanisms.

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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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