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

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

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

6. Michaelis – Menten Kinetics EnzymeSubstratesEnzyme substrates complex Product

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

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

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. 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

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 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.