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Evidence Against Stabilization of the Transition State Oxyanion by a Pka-Perturbed RNA Base in the Peptidyl Transferase Center

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Evidence Against Stabilization of the Transition State Oxyanion by a Pka-Perturbed RNA Base in the Peptidyl Transferase Center Evidence against stabilization of the transition state oxyanion by a pKa-perturbed RNA base in the peptidyl transferase center K. Mark Parnell*, Amy C. Seila†, and Scott A. Strobel*‡§ Departments of *Molecular Biophysics and Biochemistry, †Genetics, and ‡Chemistry, Yale University, 260 Whitney Avenue, New Haven, CT 06520-8114 Edited by Harry F. Noller, University of California, Santa Cruz, CA, and approved July 16, 2002 (received for review April 8, 2002) The crystal structure of the ribosomal 50S subunit from Haloarcula activity, suggesting that 23S and 5S rRNA may constitute the marismortui in complex with the transition state analog CCdA- bulk of the peptidyl transferase center (8). phosphate-puromycin (CCdApPmn) led to a mechanistic proposal The most unambiguous evidence that the active site of the wherein the universally conversed A2451 in the ribosomal active ribosome is comprised of RNA came from the 2.4-Å crystal site acts as an ‘‘oxyanion hole’’ to promote the peptidyl transferase structure of the Haloarcula marismortui 50S ribosomal subunit reaction [Nissen, P., Hansen, J., Ban, N., Moore, P.B., and Steitz, T.A. reported by Ban et al. (9) and Nissen et al. (10). The structure of the (2000) Science 289, 920–929]. In the model, close proximity (3 Å) 50S subunit complexed with the transition-state analog CCdA- between the A2451 N3 and the nonbridging phosphoramidate phosphate-puromycin (CCdApPmn) was vital to this structural oxygen of CCdApPmn suggested that the carbonyl oxyanion identification. CCdApPmn includes the minimal components of formed during the tetrahedral transition state is stabilized by both peptidyl transferase substrates (11). CCdA binds the P site, and hydrogen bonding to the protonated A2451 N3, the pKa of which puromycin binds the A site (Fig. 1). The ␣-amino group of must be perturbed substantially. We characterize the contribution puromycin is connected covalently to the 3Ј oxygen of CCdA of the putative hydrogen bond between the N3 of A2451 and the through a phosphoramidate linkage that has a tetrahedral geometry nonbridging phosphoramidate oxygen by using chemical protec- comparable in shape to the peptidyl transfer transition state (11, tion and peptidyl transfer inhibition assays. If this putative hydro- 12). Within the cocrystal structure only 23S rRNA contacts gen bond makes a significant thermodynamic contribution, then CCdApPmn. In fact, the nearest protein residue is almost 20 Å CCdApPmn-binding affinity to the 50S ribosomal subunit should be removed from the phosphoramidate linkage, which argues that strongly pH-dependent, with affinity increasing as the pH is low- rRNA and not protein catalyzes peptidyl transfer (10). ered. We report that CCdApPmn binds 50S ribosomes with essen- Within the 50S structure, the N3 of the universally conserved tially equal affinity at all pH values between 5.0 and 8.5. These data A2451 (Escherichia coli numbering) is within hydrogen-bonding argue against a mechanism for peptidyl transfer in which a residue distance (3 Å) of the nonbridging phosphoramidate oxygen with near neutral pKa stabilizes the transition-state oxyanion, at (designated O2 in the crystal coordinates) of CCdApPmn (Fig. least to the extent that CCdApPmn accurately mimics the transition 1a). The implied hydrogen bond occurs despite the fact that state. neither the O2 oxygen nor the N3 imino group would normally be protonated at pH 5.8, the pH used for crystallization of the he ribosome is a molecular machine that assembles polypep- 50S subunit (10). Based on the assumption that O2 is analogous Ttide chains. The addition of an amino acid onto a nascent to the negatively charged oxyanion formed in the transition state, peptide chain, termed peptidyl transfer, is catalyzed by the 50S the close approach of these two groups led Nissen et al. to two ribosomal subunit by using aminoacyl-tRNA and peptidyl-tRNA conclusions: (i) the N3 pKa of A2451 is perturbed toward as substrates. In the course of the reaction, the peptidyl-tRNA, neutrality in the transition state, and (ii) the protonated A2451 charged with the growing peptide chain, occupies the P site, and N3 stabilizes the negative charge on the transition state oxyanion an aminoacyl-tRNA, activated with a single amino acid, binds by hydrogen bonding and͞or charge neutralization. In this the A site. Peptide bond formation occurs by a transacylation manner A2451 was proposed to serve as the oxyanion hole for reaction mechanism wherein the ␣-amino group on the A-site the peptidyl transfer reaction (ref. 10; Fig. 1b). This contribution tRNA nucleophilically attacks the ester linkage between the is in addition to its role as a general base for activation of the peptide chain and the 3Ј-hydroxyl of the P-site tRNA. It is nucleophilic ␣-amino group. expected that the reaction proceeds through a transition state Biochemical experiments have attempted to determine the that has a tetrahedral geometry at the carbonyl carbon and extent to which the A2451 pKa is perturbed in the ground state includes a negatively charged oxyanion. Collapse of the transi- of the ribosome. The pH dependence of dimethyl sulfate (DMS) tion state produces a deacylated P-site tRNA and a peptide chain reactivity at A2451 suggested that the active-site residue has an that is elongated by one amino acid coupled to the A-site tRNA unusually high pKa of 7.6 (13). However, subsequent experi- (for review see ref. 1). ments found that DMS modification at A2451 occurred only in Defining how this reaction is catalyzed has been a question of inactive E. coli 50S subunits, and no reactivity was observed at active research for over 30 years. Despite an early and rather any pH after heat activation (14). Furthermore, the DMS indirect indication that a protein side chain might be responsible modification most likely occurred at the N1 rather than the N3 for catalysis (2, 3), biochemical evidence has identified RNA, imino group of A2451 (15). DMS protection experiments per- which accounts for about two thirds of ribosomal molecular formed on ribosomes from several different organisms showed weight (4), as the most likely catalytic component. Highly either no reactivity at A2451 or a reactivity pattern inconsistent conserved internal loops of the 23S rRNA domain V have been with a direct pKa effect (15, 16). Overall, these data are more shown biochemically to interact with the 3Ј-CCA ends of the A-site and P-site tRNAs (5, 6) as well as aminoacyl residues attached to the P-site tRNA (7). These results showed that rRNA This paper was submitted directly (Track II) to the PNAS office. is in close proximity to the nucleophile and leaving group. Other Abbreviations: CCdApPmn, CCdA-phosphate-puromycin; DMS, dimethyl sulfate; pcb, phe- experiments showed that large ribosomal subunit particles nylalanyl-caproyl-biotin; CPmn, C-puromycin. stripped of 95% of the ribosomal protein retained catalytic §To whom reprint requests should be addressed. E-mail: [email protected]. 11658–11663 ͉ PNAS ͉ September 3, 2002 ͉ vol. 99 ͉ no. 18 www.pnas.org͞cgi͞doi͞10.1073͞pnas.182210099 Downloaded by guest on October 2, 2021 be higher at acidic pH and become progressively weaker as the pH is raised. We have explored this hypothesis by measuring the pH dependence of CCdApPmn binding. The results argue against transition-state oxyanion stabilization by the peptidyl transferase center of the ribosome insofar as CCdApPmn is an accurate mimic of that transition state. Materials and Methods Synthesis of CCdApPmn. The synthesis of CCdApPmn followed that described by Welch et al. (11) with minor modifications. CCdAp (100 nmol, Dharmacon Research, Lafayette, CO) was coupled to puromycin (12.3 ␮mol) in the presence of 1-ethyl-3- [3-(dimethylamino)propyl]carbodiimide (EDAC, 50 ␮mol) buff- ered with 400 mM Mes at pH 6.0 in an aqueous reaction volume of 125 ␮l. The reaction was carried out for4hat25°C. The mixture then was diluted to 500 ␮l with water and added to a 200-␮l A-25 Sephadex column. The column was washed with 4 ml of water, followed by 4 ml of 30 mM NH4OAc (pH 6.5), and eluted with 4 ml of 750 mM NH4OAc. CCdApPmn was purified further by HPLC with a C18 column (Rainin Instruments) and ͞ eluted with a 100 mM NH4OAc (pH 6.5) acetonitrile gradient (0–50% acetonitrile) over 80 min. The retention time of CCdApPmn was 38 min. Product formation was confirmed by mass spectrometry (Howard Hughes Medical Institute͞Keck Biotechnology Resource Laboratory, Yale University, New Ha- ven, CT) and 31P NMR: theoretical m͞z, 1,393.36; actual m͞z, 1393.35. 31P NMR: ␦, Ϫ0.09, 0.14, 5.79 ppm. BIOCHEMISTRY Preparation of Ribosomes. Ribosomal 50S subunits were isolated from early log-phase MRE600 cells and prepared as described by Rheinberger et al. (17) with minor modifications. Chemical Modification of 23S rRNA. The 23S rRNA within intact ribosomal particles was modified with DMS. Each reaction contained 50 nM 50S ribosomes (preheated at 42°C for 5 min), ͞ 200 mM KCl, 20 mM MgCl2, 50 mM buffer (KOAc, pH 5.0 Mes, pH 5.5, pH 6.0͞Mops, pH 6.5, pH 7.0, pH 7.5͞Tris-Cl, pH 8.0͞Tris-Borate, pH 8.5), 33% methanol, and 0–1,500 nM CCdApPmn in a total volume of 25 ␮l. DMS was added to ribosomes (1 ␮l of a 1:10 DMS͞ethanol solution prepared Fig. 1. Peptidyl transferase transition-state inhibitor and proposed mecha- immediately before the reaction) and allowed to react at 25°C for nism of transition-state stabilization based on the inhibitor structure. (a) 30 min. The RNA was precipitated by adding 2.5 volumes of Schematic representation of CCdApPmn in the peptidyl transferase center of ethanol and stored at Ϫ80°C for 4 h, after which the ribosomes the ribosome, indicating the stereochemistry about the phosphoramidate.
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