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The Puzzling Properties of Peptidyl Transferase. Gregory W. Muth Department of Chemistry St. Olaf College. Peptidyl Transferase Reaction. Composition of the Ribosome. Proposed General Acid-Base Mechanism of Peptidyl Transferase. General Acid Catalysis. General Base Catalysis.

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slide1

The Puzzling Properties of Peptidyl Transferase

Gregory W. Muth

Department of Chemistry

St. Olaf College

slide4

Proposed General Acid-Base Mechanism

of Peptidyl Transferase

General Acid

Catalysis

General Base

Catalysis

slide5

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.”

slide7

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)

slide9

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)

slide10

pH Dependent DMS Reactivity Provides

a Reasonable Estimate of a Nucleoside’s pKa

slide12

Primer Extension

dTTP

dATP

dCTP

dGTP

Primer

32P

RT

3’

5’

RNA

Primer

32P

CH3

RT

3’

5’

RNA

STOP

STOP

STOP

slide15

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/

slide16

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
slide17

Crystal Structure of the Large

Ribosomal Subunit at 2.4 Å Resolution

Ban et. al., Science. 289, 905 (2000)

slide18

The catalytic core is composed solely of RNA

Nissen et. al., Science. 289, 920 (2000)

slide19

Crystallography

ChemicalFootprinting

Kinetics

Mechanistic

Clues

Phylogenetic

Comparison

Mutagenesis

slide20

Position of A2451 within the crystal structure shows N3

as the potential site of perturbation

Nissen, P. et al. Science (2000), 289, 920

slide21

Is the Mechanism Analogous to that of the Serine Protease Acylation Reaction?

General Base

Catalysis

General Acid

Catalysis

slide22

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?
slide23

The N3 of Adenosine is Methylated in DNA and RNA

N1

N3

P.D. Lawley & P. Brookes, Biochem. J.89, 127-138 (1963)

slide24

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)

slide25

Dimroth analysis of A2451 in E. coli ribosomes

Most consistent with modification at N1 not N3 position

slide26

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?
slide27

H. marismortui Ribosomes

DMS Modification Pattern at A2451 is pH Inverted

slide28

S. cerevisiae Ribosomes

C2452 not A2451 shows pH dependent DMS reactivity

slide29

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?
slide30

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
slide31

Noncanonical A·C pairs require

a protonated adenosine N1

C2063

slide32

Crystallography

ChemicalFootprinting

Kinetics

Mechanistic

Clues

Phylogenetic

Comparison

Mutagenesis

slide33

Kinetic Assay with Chemistry as the Rate Limiting Step

Katunin, V.I. et al, submitted for publication (2001)/

slide34

Rapid kinetics suggest more than one titratable group

Native ribosomes/puromycin

pka = 7.5 ± 0.1

m = 1.5

slide35

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
slide37

Ribosomes Can Catalyze Ester Bond Formation Using a Nucleophile with a Substantially Different pKa

Fahnestock et al. Biochemistry 12, 1970, 2477-83

slide38

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

slide39

Kinetic assay to isolate pKa2

Native ribosomes/hydroxy-puromycin

pka = 7.5 ± 0.1

m = 0.93 ± 0.05

slide40

Kinetic assay to isolate pKa1

A2451U mutant ribosomes/puromycin

pka = 6.9 ± 0.2

m  1

slide41

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

slide42

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
slide43

The Cast and Crew

Lori Ortoleva-Donnelly

Vladimir Katunin

Wolfgang Wintermeyer

Marina Rodnina

Funding: American Cancer Society (GWM)

Yale University (GWM)

NIH, NSF (SAS)

slide45

On the next exciting episode…

  • Unraveling the mysteries of RNA folding
slide46

RNA motifs

tetraloop

K-turn