Slide 1

The Puzzling Properties of Peptidyl Transferase Gregory W. Muth Department of Chemistry St. Olaf College Slide 2 Peptidyl Transferase Reaction Slide 3 Composition of the Ribosome Slide 4 Proposed General Acid-Base Mechanism of Peptidyl Transferase General Base Catalysis General Acid Catalysis Slide 5 B.E. H. Maden & R. E. Monro, European J. Biochem. 6, 309-316 (1968) Isolated 50S ribosomes pK a = 7.5-8.0 S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 69, 624-628 (1972) Polyribosomes pK a = 7.2-7.4 The pK a of the PTase Reaction is Between 7.2 and 8.0 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. 7.367.247.2 Slide 6 RNA Lacks Functional Groups with a Neutral pK a Slide 7 The Tertiary Fold of an RNA Strand can change the pK a of the Bases Structural Examples:Catalytic Example: C + G-C Triple (pK a = 7.2) A + C Pair (pK a = 6.6) C75 in the HDV Ribozyme (pK a = 5.7) Ferre-D Amare, Zhou & Doudna, Nature 395, 567-74 (1998) Nakano, Chadalavada, Bevilacqua, Science 287, 1493-97 (2000) Slide 8 pH Dependent DMS Modification : Determination of a Nucleotides pK a Slide 9 c 3 A pK a from Minakawa, Kojima & Matsuda, J. Org. Chem. 64, 7158-72 (1999) Model System: pK a Determination of c 3 A by pH Dependent DMS Reactivity Methylation of 3-deaza-adenosine as a function of time Slide 10 pH Dependent DMS Reactivity Provides a Reasonable Estimate of a Nucleosides pK a Slide 11 Secondary Structure of 23S rRNA Slide 12 Primer Extension RT Primer RNA 55 33 Primer RNA 55 33 dATP dTTP dCTP dGTP CH 3 STOP 32 P RT Slide 13 DMS Mapping of Domain V within 50S Ribosomal Subunits as a Function of pH * Slide 14 The pKa of A2451 is Apparently Perturbed Above Neutrality Slide 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, Cell 57, 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, Biochimie 69, 879-884 (1987) R. Gutell, et al., http://www.rna.icmb.utexas.edu/ Slide 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 P L promoter The mutant plasmids were transformed into E. coli pop2136 cells which express a temperature sensitive form of repressor Slide 17 Ban et. al., Science. 289, 905 (2000) Crystal Structure of the Large Ribosomal Subunit at 2.4 Resolution Slide 18 The catalytic core is composed solely of RNA Nissen et. al., Science. 289, 920 (2000) Slide 19 Mechanistic Clues CrystallographyChemical Footprinting Kinetics Mutagenesis Phylogenetic Comparison Slide 20 Nissen, P. et al. Science (2000), 289, 920 Position of A2451 within the crystal structure shows N3 as the potential site of perturbation Slide 21 General Base Catalysis General Acid Catalysis Is the Mechanism Analogous to that of the Serine Protease Acylation Reaction? Slide 22 Further experiments to refine the A2451 pK a interpretation: 1. Determine the specificity of methylation: N1 vs N3 2. Is the pK a perturbation conserved across phylogeny? 3. Is there another titratable group with a pKa near neutral? Slide 23 The N3 of Adenosine is Methylated in DNA and RNA N1 N3 P.D. Lawley & P. Brookes, Biochem. J. 89, 127-138 (1963) Slide 24 Distinguishing N1 from N3 Methylation by Dimroth Rearrangement upon Alkaline pH Incubation Macon and Wolfenden, Biochemistry 7, 3453-58 (1968) Saito and Fujii, J. Chem. Soc. Chem. Comm. 1979, 135 (1979) Slide 25 Dimroth analysis of A2451 in E. coli ribosomes Most consistent with modification at N1 not N3 position Slide 26 Further experiments to refine the A2451 pK a interpretation: 1. Determine the specificity of methylation: N1 vs N3 2. Is the pK a perturbation conserved across phylogeny? 3. Is there another titratable group with a pK a near neutral? Slide 27 H. marismortui Ribosomes DMS Modification Pattern at A2451 is pH Inverted Slide 28 S. cerevisiae Ribosomes C2452 not A2451 shows pH dependent DMS reactivity Slide 29 Further experiments to refine the A2451 pK a interpretation: 1. Determine the specificity of methylation: N1 vs N3 2. Is the pK a perturbation conserved across phylogeny? 3. Is there another titratable group with a pK a near neutral? Slide 30 A2451 is Flanked by Two Noncanonical AC Pairs The A2450C2063 pair is highly conserved and has a wobble geometry The A2453C2499 pair is less well conserved and has a wobble-like geometry Slide 31 Noncanonical AC pairs require a protonated adenosine N1 C2063 Slide 32 Mechanistic Clues CrystallographyChemical Footprinting Kinetics Mutagenesis Phylogenetic Comparison Slide 33 Kinetic Assay with Chemistry as the Rate Limiting Step Katunin, V.I. et al, submitted for publication (2001) / Slide 34 Native ribosomes/puromycin pk a = 7.5 0.1 m = 1.5 Rapid kinetics suggest more than one titratable group Slide 35 Nuc-H + Ribosome-H + Nuc RibosomeNuc Ribosome-H + pK a1 pK a2 Model for Protonation Events within the Ribosome Measue pK a of puromycin Replace nitrogen nucleophile with hydroxyl Mutate active site residue Slide 36 Puromycin pk a = 6.9 0.2 pK a of the nucleophile is below that of the reaction Slide 37 Ribosomes Can Catalyze Ester Bond Formation Using a Nucleophile with a Substantially Different pK a Fahnestock et al. Biochemistry 12, 1970, 2477-83 Slide 38 Synthesis of Hydroxy-purmomycin i) TMS-Cl, pyridine ii) TBDMS-Cl, imidizole, DMF iii) oxalyl chloride, CH 2 Cl 2, DMF (cat.) iv) addition of nucleoside to excess acylchloride, quench with NH 4 OH/H 2 O v) TBAF, THF Slide 39 pk a = 7.5 0.1 Native ribosomes/hydroxy-puromycin m = 0.93 0.05 Kinetic assay to isolate pK a2 Slide 40 A2451U mutant ribosomes/puromycin pk a = 6.9 0.2 m 1 Kinetic assay to isolate pK a1 Slide 41 Puromycin-H + A2451-H + Puromycin A2451Puromycin A2451-H + pK a1 pK a2 6.97.5 General Base Catalysis General Acid Catalysis Does A2451 hold chemical or structural importance? Slide 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 pK a = 7.5 Chemical footprinting suggests a pH dependent, active site conformational change, possibly due to two highly conserved A-C pairs Slide 43 The Cast and Crew Funding: American Cancer Society (GWM) Yale University (GWM) NIH, NSF (SAS) Lori Ortoleva-Donnelly Vladimir Katunin Wolfgang Wintermeyer Marina Rodnina Slide 44 Slide 45 On the next exciting episode Unraveling the mysteries of RNA folding Slide 46 RNA motifs tetraloopK-turn Slide 47