Regiospecificity of the peptidyl tRNA ester within the ribosomal P site

Article abstract of DOI:10.1021/ja0554099

The ribosomal peptidyl transferase center is expected to be regiospecific with regard to its tRNA substrates, yet the ester linkages between the tRNA and the amino acid or peptide are susceptible to isomerization between the O2′ and O3′ hydroxyls of the t

Full text of DOI:10.1021/ja0554099

Published on Web 02/14/2006
Regiospecificity of the Peptidyl tRNA Ester within the Ribosomal P Site
Kevin S. Huang, Joshua S. Weinger, Ethan B. Butler, and Scott A. Strobel*
Department of Molecular Biophysics and Biochemistry, Yale UniVersity, 260 Whitney AVenue,
New HaVen, Connecticut 06520-8114
Received August 8, 2005; E-mail: scott.strobel@yale.edu
The ribosome utilizes two substrates during polypeptide synthe-
sis: an aminoacyl tRNA in the A site and a peptidyl tRNA in the
P site. Peptide bond formation involves aminolysis of the P site
ester by the A-site amino acid. Both the peptide and amino acid
are linked to their tRNAs through an ester bond to A76, the 3′-
terminal residue of each tRNA. The reaction is expected to be
regiospecific with regard to its tRNA substrates, yet the ester
linkages between the tRNA and the amino acid or peptide are
susceptible to isomerization between the O2′ and O3′ hydroxyls
of the terminal A76 ribose sugar. The uncatalyzed rate of amino
acid isomerization in aqueous solution is approximately 5 s-1, and
the thermodynamic equilibrium between the two isomers is ap-
proximately 1.1 The reaction products are the same beginning with
either an O2′- or O3′-linked mechanism, but the geometry of the
nucleophile, the leaving group, and the oxyanion are substantially
different for reaction of the two regioisomers.
Figure 1. Two distinct pathways for the ribosome-catalyzed peptide bond
formation. (a) Isomerization of the peptidyl ester on the terminal A76 of
the P-site tRNA leads to two different intermediates linked via the O2′
(right) or the O3′ (left) oxygen. (b) Nonisomerizable mimics of these
intermediates feature C74, C75, and A76 of the peptidyl and aminoacyl
tRNA acceptor ends. These inhibitors also contain a phosphorus atom to
mimic the tetrahedral carbon formed in the intermediate.
The regiospecificity for the A-site substrate has been established
as the O3′ isomer.2-4 tRNA analogues that force the amino acid
onto the O3′ position, such as puromycin (Pm) and 2′-deoxyad-
enosine (2′-dA), are active as peptide acceptors. Similar analogues
that force the amino acid to the O2′ are inactive. The A-site product
following peptidyl transfer is also O3′-linked, consistent with this
regiospecificity.5
We prepared two regioisomers of the peptidyl transferase
inhibitor, CCApPmCC, where the phosphodiester is either O3′- or
O2′-linked to the P-site A76 (Figure 1b). These analogues were
prepared by solid phase chemical synthesis based upon the method
described previously.13 They include the critical A76 hydroxyl
vicinal to the phosphodiester linkage.9 Recent structural studies on
A-site substrates indicated that the peptidyl transferase center (PTC)
is induced into an active conformation by the stacking of the A-site
nucleotide C74.14 To ensure that the ribosome is in the induced
state, both isomers include C74 and C75 on both the P-site and
A-site segment. Most importantly, and unlike substrates previously
used to explore this question, these compounds cannot undergo
regioisomerization.
Transition state theory predicts that the ribosomal PTC will bind
tighter to the transition state than to the substrate or product states
of the reaction. Consequently, the compound with greatest affinity
for the PTC is indicative of the tetrahedral intermediate’s re-
giospecificity. The affinities of the O2′ and O3′ analogues were
determined by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
metho-p-toluenesulfonate (CMCT) chemical footprinting of active
site residue U2585, as previously described.12,13,15 The extent of
CMCT modification as a function of analogue concentration was
determined by reverse transcription, and the resulting data were
used to calculate the binding constant (Figure 2). The O3′-linked
inhibitor bound the ribosome with an affinity of 280 ( 40 nM
(Figure 2a). This is 7-fold tighter than that observed for the original
CCdApPmn inhibitor under the same conditions (data not shown),
consistent with the additional stacking and base pairing interactions
by C74 and C75 in the A site. The O2′ analogue showed only slight
binding at concentrations greater than 10 µM (Figure 2b). This is
In contrast to the A site, the regiospecificity of the peptidyl tRNA
in the P site has not been determined (Figure 1a).2 An early report
suggested that a tRNA, which had a 2′-dA substitution at position
76, was active as a peptide donor, while the O2′-linked 3′-dA76
substitution was inactive.6 This led to the widely held conclusion
that the P-site ester is O3′-linked (Figure 1a, left); however, all
other studies on P-site substrates lacking either the 2′- or 3′-OH
demonstrated that both are inactive as peptide donors.7-9 Conse-
quently, the functional regioisomer cannot be assigned from such
studies. A cocrystal structure of a peptidyl tRNA fragment in the
P-site and sparsomycin in the A site suggests that the ribosome
binds preferentially to the O3′-linked regioisomer.10 While this is
consistent with reaction via the O3′-linked intermediate, it remains
possible that the ribosome catalyzes peptide migration to the O2′
position in the course of the reaction, resulting in an O2′-linked
transition state (Figure 1a, right). Potentially consistent with this
possibility is the structure of the 50S ribosome in complex with
the transition state inhibitor CCdApPm.11 This compound includes
a phosphoramidate to mimic the putative tetrahedral intermediate,
but lacked a 2′-OH at the P-site A76.12 In the cocrystal structure,
the nonbridging phosphoramidate oxygen was closer to the C2′
carbon than would be possible if the 2′-OH were present. Such a
conformation raises the possibility that the ribosome may prefer to
bind a transition state with an O2′ linkage to the P-site tRNA. The
ambiguous biochemical and structural data prompted us to explore
an alternative approach to establish the regiospecificity of the
reaction.
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10.1021/ja0554099 CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
The marked preference for the O3′ isomer suggests that the
ribosome uses the O3′-linked tRNA as the P-site substrate. Because
this is the same regioisomer used in the A-site, it indicates that the
aminoacyl tRNA ester is not required to undergo regioisomerization
during its course through the PTC.
Preferential binding and inhibition by the O3′-linked isomer
clarifies an important ambiguity in the previous biochemical data
regarding the inactivity of 2′- or 3′-dA76 P-site substrates.2,6-9 The
current data argue that substrates lacking a 3′-OH are inactive as
donors because they are constrained into the wrong O2′-linked
regioisomer. In contrast, P-site substrates lacking the 2′-OH adopt
the correct O3′-linked regioisomer, but are inactive as P-site donors
because the 2′-OH plays a critical role in substrate assisted catalysis
of the peptidyl transferase reaction.9 The current data exclude a
mechanistic model in which the A76 2′-OH participates as a
covalent intermediate in the reaction.
In conclusion, we have established the regiospecificity of the
ribosomal peptidyl transferase reaction. We are now in the process
of utilizing these inhibitors to understand the role of the critical
A76 2′-OH and to probe the stereospecificity and potential metal
ion dependence of peptide bond formation.
Figure 2. Regiospecificity of analogue binding by chemical footprinting
and peptidyl transferase inhibition assays. (a) CMCT footprinting of U2585
in the PTC at 37 °C. Shown is an autoradiograph of primer extension
reaction from CMCT modification of U2585 in the presence of the O3′ or
the O2′ analogue. Concentrations and presence of CMCT are indicated above
each lane. Bands for U2585 and U2477, which is used to normalize the
footprinting data, are marked with an arrow. (b) Plot of footprinting data
used to estimate the relative dissociation constants (Kd).
Acknowledgment. We thank Olke Uhlenbeck and Jesse Co-
chrane for helpful discussions, and David E. Kitchen (Dharmacon
Research Inc.) and Olga Fedorova for technical assistance in solid
phase synthesis. This work was supported by a an American Cancer
Society Beginning Investigator award to S.A.S. and a National
Institutes of Health Postdoctoral Fellowship to K.S.H.
Supporting Information Available: Synthetic, CMCT modifica-
tion, and kinetic procedures are included. This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 3. Inhibition of the modified fragment reaction. (a) Sample reaction
time courses performed at pH 7.0 with 100 nM E. coli 50S ribosomes and
400 nM CCApcb. The reactions shown had 200 µM CPuromycin and no
inhibitor (O), 300 nM O3′ inhibitor (0), or 300 nM O2′ inhibitor ()).
Background hydrolysis in the absence of CPuromycin is also shown (×).
(B) Ki values for the inhibitors were determined by varying the CPuromycin
concentration from 20 to 400 µM.
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