Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


Presentation on theme: "The Puzzling Properties of Peptidyl Transferase Gregory W. Muth Department of Chemistry St. Olaf College."— Presentation transcript:

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 Base Catalysis General Acid Catalysis

5 B.E. H. Maden & R. E. Monro, European J. Biochem. 6, (1968) Isolated 50S ribosomes pK a = S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 69, (1972) Polyribosomes pK a = 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.”

6 RNA Lacks Functional Groups with a Neutral pK a

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, (1998) Nakano, Chadalavada, Bevilacqua, Science 287, (2000)

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

9 c 3 A pK a from Minakawa, Kojima & Matsuda, J. Org. Chem. 64, (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

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

11 Secondary Structure of 23S rRNA

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

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, Cell 57, (1989) A2451 is cross-linked with a P-site bound t-RNA Steiner, Kuechler & Barta, EMBO J. 7, (1988) A2451is footprinted by peptidyl transferase inhibiting antibiotics Moazed & Noller, Biochimie 69, (1987) R. Gutell, et al.,

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

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

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

19 Mechanistic Clues CrystallographyChemical Footprinting Kinetics Mutagenesis Phylogenetic Comparison

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

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

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?

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

24 Distinguishing N1 from N3 Methylation by Dimroth Rearrangement upon Alkaline pH Incubation Macon and Wolfenden, Biochemistry 7, (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 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?

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 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?

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 Mechanistic Clues CrystallographyChemical Footprinting Kinetics Mutagenesis Phylogenetic Comparison

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

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

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

36 Puromycin pk a = 6.9 ± 0.2 pK a of the nucleophile is below that of the reaction

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

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

39 pk a = 7.5 ± 0.1 Native ribosomes/hydroxy-puromycin m = 0.93 ± 0.05 Kinetic assay to isolate pK a2

40 A2451U mutant ribosomes/puromycin pk a = 6.9 ± 0.2 m  1 Kinetic assay to isolate pK a1

41 Puromycin-H +  A2451-H + Puromycin  A2451Puromycin  A2451-H + pK a1 pK a General Base Catalysis General Acid Catalysis Does A2451 hold chemical or structural importance?

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

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

44

45 On the next exciting episode… Unraveling the mysteries of RNA folding

46 RNA motifs tetraloopK-turn

47


Download ppt "The Puzzling Properties of Peptidyl Transferase Gregory W. Muth Department of Chemistry St. Olaf College."

Similar presentations


Ads by Google