The Puzzling Properties of Peptidyl Transferase

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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.
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RNA Lacks Functional Groups with a Neutral pKa
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The Tertiary Fold of an RNA Strand can change the
pKa of the Bases
Structural Examples
Catalytic Example
CG-C Triple (pKa 7.2)
C75 in the HDV Ribozyme (pKa 5.7)
Ferre-D Amare, Zhou Doudna, Nature 395, 567-74
(1998) Nakano, Chadalavada, Bevilacqua, Science
287, 1493-97 (2000)
AC Pair (pKa 6.6)
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pH Dependent DMS Modification Determination of
a Nucleotides pKa
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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)
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pH Dependent DMS Reactivity Provides a
Reasonable Estimate of a Nucleosides pKa
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Secondary Structure of 23S rRNA
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Primer Extension
dTTP
dATP
dCTP
dGTP
Primer
32P
RT
5
3
RNA
Primer
32P
CH3
RT
5
3
RNA
STOP
STOP
STOP
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DMS Mapping of Domain V within 50S Ribosomal
Subunits as a Function of pH

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The pKa of A2451 is Apparently Perturbed Above
Neutrality
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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/
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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

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Crystal Structure of the Large Ribosomal Subunit
at 2.4 Å Resolution
Ban et. al., Science. 289, 905 (2000)
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The catalytic core is composed solely of RNA
Nissen et. al., Science. 289, 920 (2000)
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Crystallography
Chemical Footprinting
Kinetics
Mechanistic Clues
Phylogenetic Comparison
Mutagenesis
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Position of A2451 within the crystal structure
shows N3 as the potential site of perturbation
Nissen, P. et al. Science (2000), 289, 920
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Is the Mechanism Analogous to that of the Serine
Protease Acylation Reaction?
General Base Catalysis
General Acid Catalysis
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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?
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The N3 of Adenosine is Methylated in DNA and RNA
N1
N3
P.D. Lawley P. Brookes, Biochem. J. 89, 127-138
(1963)
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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)
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Dimroth analysis of A2451 in E. coli ribosomes
Most consistent with modification at N1 not N3
position
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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?
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H. marismortui Ribosomes DMS Modification Pattern
at A2451 is pH Inverted
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S. cerevisiae Ribosomes C2452 not A2451 shows pH
dependent DMS reactivity
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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?
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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

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Noncanonical AC pairs require a protonated
adenosine N1
C2063
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Crystallography
Chemical Footprinting
Kinetics
Mechanistic Clues
Phylogenetic Comparison
Mutagenesis
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Kinetic Assay with Chemistry as the Rate Limiting
Step
Katunin, V.I. et al, submitted for publication
(2001)/
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Rapid kinetics suggest more than one titratable
group
Native ribosomes/puromycin
pka 7.5 0.1
m 1.5
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Model for Protonation Events within the Ribosome
pKa1
pKa2
Nuc-H?Ribosome-H
Nuc?Ribosome
Nuc?Ribosome-H

• Measue pKa of puromycin
• Replace nitrogen nucleophile with hydroxyl
• Mutate active site residue

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pKa of the nucleophile is below that of the
reaction
Puromycin
pka 6.9 0.2
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Ribosomes Can Catalyze Ester Bond Formation Using
a Nucleophile with a Substantially Different pKa
Fahnestock et al. Biochemistry 12, 1970, 2477-83
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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
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Kinetic assay to isolate pKa2
Native ribosomes/hydroxy-puromycin
pka 7.5 0.1
m 0.93 0.05
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Kinetic assay to isolate pKa1
A2451U mutant ribosomes/puromycin
pka 6.9 0.2
m ? 1
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Does A2451 hold chemical or structural importance?
pKa1
pKa2
Puromycin-H?A2451-H
Puromycin?A2451
Puromycin?A2451-H
6.9
7.5
General Base Catalysis
General Acid Catalysis
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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

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The Cast and Crew
Funding American Cancer Society (GWM) Yale
University (GWM) NIH, NSF (SAS)
Lori Ortoleva-Donnelly Vladimir Katunin Wolfgang
Wintermeyer Marina Rodnina
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On the next exciting episode

• Unraveling the mysteries of RNA folding

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RNA motifs
tetraloop
K-turn
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