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The Puzzling Properties of Peptidyl Transferase PowerPoint Presentation
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The Puzzling Properties of Peptidyl Transferase

The Puzzling Properties of Peptidyl Transferase

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The Puzzling Properties of Peptidyl Transferase

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  1. The Puzzling Properties of Peptidyl Transferase Gregory W. Muth Department of Chemistry St. Olaf College

  2. Peptidyl Transferase Reaction

  3. Composition of the Ribosome

  4. Proposed General Acid-Base Mechanism of Peptidyl Transferase General Acid Catalysis General Base Catalysis

  5. The pKa of the PTase Reaction is Between 7.2 and 8.0 pKa = 7.5-8.0 Isolated 50S ribosomes B.E. H. Maden & R. E. Monro, European J. Biochem. 6, 309-316 (1968) pKa = 7.2-7.4 7.36 7.24 7.2 Polyribosomes S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 69, 624-628 (1972) “The pKa of 7.2 is consistent with the possibility of a single imidazole residue being involved at the active center of the transpeptidase complex.”

  6. RNA Lacks Functional Groups with a Neutral pKa

  7. The Tertiary Fold of an RNA Strand can change the pKa of the Bases Structural Examples: Catalytic Example: C+G-C Triple (pKa = 7.2) C75 in the HDV Ribozyme (pKa = 5.7) Ferre-D’ Amare, Zhou & Doudna, Nature395, 567-74 (1998) Nakano, Chadalavada, Bevilacqua, Science287, 1493-97 (2000) A+C Pair (pKa = 6.6)

  8. pH Dependent DMS Modification : Determination of a Nucleotide’s pKa

  9. Model System: pKa Determination of c3A by pH Dependent DMS Reactivity Methylation of 3-deaza-adenosine as a function of time c3A pKa from Minakawa, Kojima & Matsuda, J. Org. Chem.64, 7158-72 (1999)

  10. pH Dependent DMS Reactivity Provides a Reasonable Estimate of a Nucleoside’s pKa

  11. Secondary Structure of 23S rRNA

  12. Primer Extension dTTP dATP dCTP dGTP Primer 32P RT 3’ 5’ RNA Primer 32P CH3 RT 3’ 5’ RNA STOP STOP STOP

  13. DMS Mapping of Domain V within 50S Ribosomal Subunits as a Function of pH *

  14. The pKa of A2451 is Apparently Perturbed Above Neutrality

  15. A2451 is Universally Conserved Several lines of experimental evidence place A2451 within the peptidyl transferase center • A2451 is DMS footprinted with a peptidyl-tRNA • Moazed & Noller, Cell57, 585-597 (1989) • A2451 is cross-linked with a P-site bound t-RNA • Steiner, Kuechler & Barta, EMBO J.7, 3949-55 (1988) • A2451is footprinted by peptidyl transferase inhibiting antibiotics • Moazed & Noller, Biochimie69, 879-884 (1987) R. Gutell, et al., http://www.rna.icmb.utexas.edu/

  16. A2451 is essential for ribosomal function in vivo • A2451 was mutated to G, C, U in the plasmid pLK35 which contains the • rrnB operon under control of the bactereophage  PL promoter • The mutant plasmids were transformed into E. coli pop2136 cells which • express a temperature sensitive form of  repressor

  17. Crystal Structure of the Large Ribosomal Subunit at 2.4 Å Resolution Ban et. al., Science. 289, 905 (2000)

  18. The catalytic core is composed solely of RNA Nissen et. al., Science. 289, 920 (2000)

  19. Crystallography ChemicalFootprinting Kinetics Mechanistic Clues Phylogenetic Comparison Mutagenesis

  20. Position of A2451 within the crystal structure shows N3 as the potential site of perturbation Nissen, P. et al. Science (2000), 289, 920

  21. Is the Mechanism Analogous to that of the Serine Protease Acylation Reaction? General Base Catalysis General Acid Catalysis

  22. Further experiments to refine the • A2451 pKa interpretation: • 1. Determine the specificity of methylation: N1 vs N3 • 2. Is the pKa perturbation conserved across phylogeny? • 3. Is there another titratable group with a pKa near neutral?

  23. The N3 of Adenosine is Methylated in DNA and RNA N1 N3 P.D. Lawley & P. Brookes, Biochem. J.89, 127-138 (1963)

  24. Distinguishing N1 from N3 Methylation by Dimroth Rearrangement upon Alkaline pH Incubation Macon and Wolfenden, Biochemistry7, 3453-58 (1968) Saito and Fujii, J. Chem. Soc. Chem. Comm.1979, 135 (1979)

  25. Dimroth analysis of A2451 in E. coli ribosomes Most consistent with modification at N1 not N3 position

  26. Further experiments to refine the • A2451 pKa interpretation: • 1. Determine the specificity of methylation: N1 vs N3 • 2. Is the pKa perturbation conserved across phylogeny? • 3. Is there another titratable group with a pKa near neutral?

  27. H. marismortui Ribosomes DMS Modification Pattern at A2451 is pH Inverted

  28. S. cerevisiae Ribosomes C2452 not A2451 shows pH dependent DMS reactivity

  29. Further experiments to refine the • A2451 pKa interpretation: • 1. Determine the specificity of methylation: N1 vs N3 • 2. Is the pKa perturbation conserved across phylogeny? • 3. Is there another titratable group with a pKa near neutral?

  30. A2451 is Flanked by Two Noncanonical A·C Pairs • The A2450·C2063 pair is highly conserved and has a wobble geometry • The A2453·C2499 pair is less well conserved and has a wobble-like geometry

  31. Noncanonical A·C pairs require a protonated adenosine N1 C2063

  32. Crystallography ChemicalFootprinting Kinetics Mechanistic Clues Phylogenetic Comparison Mutagenesis

  33. Kinetic Assay with Chemistry as the Rate Limiting Step Katunin, V.I. et al, submitted for publication (2001)/

  34. Rapid kinetics suggest more than one titratable group Native ribosomes/puromycin pka = 7.5 ± 0.1 m = 1.5

  35. Model for Protonation Events within the Ribosome pKa1 pKa2 Nuc-H+Ribosome-H+ NucRibosome-H+ NucRibosome • Measue pKa of puromycin • Replace nitrogen nucleophile with hydroxyl • Mutate active site residue

  36. pKa of the nucleophile is below that of the reaction Puromycin pka = 6.9 ± 0.2

  37. Ribosomes Can Catalyze Ester Bond Formation Using a Nucleophile with a Substantially Different pKa Fahnestock et al. Biochemistry 12, 1970, 2477-83

  38. Synthesis of Hydroxy-purmomycin i) TMS-Cl, pyridine ii) TBDMS-Cl, imidizole, DMF iii) oxalyl chloride, CH2Cl2, DMF (cat.) iv) addition of nucleoside to excess acylchloride, quench with NH4OH/H2O v) TBAF, THF

  39. Kinetic assay to isolate pKa2 Native ribosomes/hydroxy-puromycin pka = 7.5 ± 0.1 m = 0.93 ± 0.05

  40. Kinetic assay to isolate pKa1 A2451U mutant ribosomes/puromycin pka = 6.9 ± 0.2 m  1

  41. DoesA2451 hold chemical or structural importance? pKa1 pKa2 Puromycin-H+A2451-H+ PuromycinA2451-H+ PuromycinA2451 6.9 7.5 General Base Catalysis General Acid Catalysis

  42. Mechanistic Possibilities • Kinetic assays reveal potentially two titratable protons within the active site; one from the nitrogen nucleophile, the second from a ribosomal residue, supposedly A2451 • Both the kinetic assay and chemical footprinting analysis measured the ribosomal pKa = 7.5 • Chemical footprinting suggests a pH dependent, active site conformational change, possibly due to two highly conserved A-C pairs

  43. The Cast and Crew Lori Ortoleva-Donnelly Vladimir Katunin Wolfgang Wintermeyer Marina Rodnina Funding: American Cancer Society (GWM) Yale University (GWM) NIH, NSF (SAS)

  44. On the next exciting episode… • Unraveling the mysteries of RNA folding

  45. RNA motifs tetraloop K-turn