Ribosomes catalyze the formation of peptide bonds between aminoacyl esters of transfer RNAs within a catalytic center composed of ribosomal RNA only. Here we show that the reaction of P-site formylmethionine (fMet)-tRNAfMet with a modified A-site tRNA substrate, Phelac-tRNAPhe, in which the nucleophilic amino group is replaced with a hydroxyl group, does not show the pH dependence observed with small substrate analogs such as puromycin and hydroxypuromycin. This indicates that acid-base catalysis by ribosomal residues is not important in the reaction with the full-size substrate. Rather, the ribosome catalyzes peptide bond formation by positioning the tRNAs, or their 3′ termini, through interactions with rRNA that induce and/or stabilize a pH-insensitive conformation of the active site and provide a preorganized environment facilitating the reaction. The rate of peptide bond formation with unmodified Phe-tRNAPhe is estimated to be >300 s−1.
Your institute does not have access to this article
Open Access articles citing this article.
BMC Bioinformatics Open Access 30 November 2011
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Noller, H.F., Hoffarth, V. & Zimniak, L. Unusual resistance of peptidyl transferase to protein extraction procedures. Science 256, 1416–1419 (1992).
Ban, N., Nissen, P., Hansen, J., Moore, P.B. & Steitz, T.A. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289, 905–920 (2000).
Nissen, P., Hansen, J., Ban, N., Moore, P.B. & Steitz, T.A. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930 (2000).
Harms, J. et al. High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107, 679–688 (2001).
Satterthwait, A.C. & Jencks, W.P. The mechanism of the aminolysis of acetate esters. J. Am. Chem. Soc. 96, 7018–7031 (1974).
Bevilacqua, P.C., Brown, T.S., Nakano, S. & Yajima, R. Catalytic roles for proton transfer and protonation in ribozymes. Biopolymers 73, 90–109 (2004).
Muth, G.W., Chen, L., Kosek, A.B. & Strobel, S.A. pH-dependent conformational flexibility within the ribosomal peptidyl transferase center. RNA 7, 1403–1415 (2001).
Krayevsky, A.A. & Kukhanova, M.K. The peptidyltransferase center of ribosomes. Prog. Nucleic Acid Res. Mol. Biol. 23, 1–51 (1979).
Nierhaus, K.H., Schulze, H. & Cooperman, B.S. Molecular mechanisms of the ribosomal peptidyltransferase center. Biochem. Int. 1, 185–192 (1980).
Schmeing, T.M., Huang, K.S., Kitchen, D.E., Strobel, S.A. & Steitz, T.A. Structural insights into the roles of water and the 2′ hydroxyl of the P site tRNA in the peptidyl transferase reaction. Mol. Cell 20, 437–448 (2005).
Sharma, P.K., Xiang, Y., Kato, M. & Warshel, A. What are the roles of substrate-assisted catalysis and proximity effects in peptide bond formation by the ribosome? Biochemistry 44, 11307–11314 (2005).
Trobro, S. & Aqvist, J. Mechanism of peptide bond synthesis on the ribosome. Proc. Natl. Acad. Sci. USA 102, 12395–12400 (2005).
Seila, A.C., Okuda, K., Nunez, S., Seila, A.F. & Strobel, S.A. Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction. Biochemistry 44, 4018–4027 (2005).
Sievers, A., Beringer, M., Rodnina, M.V. & Wolfenden, R. The ribosome as an entropy trap. Proc. Natl. Acad. Sci. USA 101, 7897–7901 (2004).
Hansen, J.L., Schmeing, T.M., Moore, P.B. & Steitz, T.A. Structural insights into peptide bond formation. Proc. Natl. Acad. Sci. USA 99, 11670–11675 (2002).
Yusupov, M.M. et al. Crystal structure of the ribosome at 5.5 Å resolution. Science 292, 883–896 (2001).
Bashan, A. et al. Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression. Mol. Cell 11, 91–102 (2003).
Kim, D.F. & Green, R. Base-pairing between 23S rRNA and tRNA in the ribosomal A site. Mol. Cell 4, 859–864 (1999).
Pape, T., Wintermeyer, W. & Rodnina, M.V. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E.coli ribosome. EMBO J. 17, 7490–7497 (1998).
Katunin, V.I., Muth, G.W., Strobel, S.A., Wintermeyer, W. & Rodnina, M.V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome. Mol. Cell 10, 339–346 (2002).
Youngman, E.M., Brunelle, J.L., Kochaniak, A.B. & Green, R. The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release. Cell 117, 589–599 (2004).
Wolfenden, R. The mechanism of hydrolysis of amino acyl RNA. Biochemistry 338, 1090–1092 (1963).
Fahnestock, S. & Rich, A. Synthesis by ribosomes of viral coat protein containing ester linkages. Nat. New Biol. 229, 8–10 (1971).
Fahnestock, S., Neumann, H., Shashoua, V. & Rich, A. Ribosome-catalyzed ester formation. Biochemistry 9, 2477–2483 (1970).
Schmeing, T.M., Huang, K.S., Strobel, S.A. & Steitz, T.A. An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA. Nature 438, 520–524 (2005).
Derwenskus, K.H. & Sprinzl, M. Interaction of cinnamyl-tRNAPhe with Escherichia coli elongation factor Tu. FEBS Lett. 151, 143–147 (1983).
Moazed, D. & Noller, H.F. Intermediate states in the movement of transfer RNA in the ribosome. Nature 342, 142–148 (1989).
Bevilacqua, P.C. Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme. Biochemistry 42, 2259–2265 (2003).
Fedor, M.J. & Williamson, J.R. The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6, 399–412 (2005).
Okuda, K., Seila, A.C. & Strobel, S.A. Uncovering the enzymatic pKa of the ribosomal peptidyl transferase reaction utilizing a fluorinated puromycin derivative. Biochemistry 44, 6675–6684 (2005).
Muth, G.W., Ortoleva-Donnelly, L. & Strobel, S.A. A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center. Science 289, 947–950 (2000).
Xiong, L., Polacek, N., Sander, P., Bottger, E.C. & Mankin, A. pKa of adenine 2451 in the ribosomal peptidyl transferase center remains elusive. RNA 7, 1365–1369 (2001).
Bayfield, M.A., Dahlberg, A.E., Schulmeister, U., Dorner, S. & Barta, A. A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition. Proc. Natl. Acad. Sci. USA 98, 10096–10101 (2001).
Brunelle, J.L., Youngman, E.M., Sharma, D. & Green, R. The interaction between C75 of tRNA and the A loop of the ribosome stimulates peptidyl transferase activity. RNA 12, 33–39 (2006).
Hesslein, A.E. et al. Exploration of the conserved A+C wobble pair within the ribosomal peptidyl transferase center using affinity purified mutant ribosomes. Nucleic Acids Res. 32, 3760–3770 (2004).
Beringer, M. et al. Essential mechanisms in the catalysis of peptide bond formation on the ribosome. J. Biol. Chem. 280, 36065–36072 (2005).
Weinger, J.S., Parnell, K.M., Dorner, S., Green, R. & Strobel, S.A. Substrate-assisted catalysis of peptide bond formation by the ribosome. Nat. Struct. Mol. Biol. 11, 1101–1106 (2004).
Das, G.K., Bhattacharyya, D. & Burma, D.P. A possible mechanism of peptide bond formation on ribosome without mediation of peptidyl transferase. J. Theor. Biol. 200, 193–205 (1999).
Radzicka, A. & Wolfenden, R. A proficient enzyme. Science 267, 90–93 (1995).
Rodnina, M.V. & Wintermeyer, W. Fidelity of aminoacyl-tRNA selection on the ribosome: kinetic and structural mechanisms. Annu. Rev. Biochem. 70, 415–435 (2001).
Rodnina, M.V. & Wintermeyer, W. GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs. Proc. Natl. Acad. Sci. USA 92, 1945–1949 (1995).
Rodnina, M.V. et al. Thiostrepton inhibits turnover but not GTP hydrolysis by elongation factor G on the ribosome. Proc. Natl. Acad. Sci. USA 96, 9586–9590 (1999).
Johnson, A.E., Adkins, H.J., Matthews, E.A. & Cantor, C.R. Distance moved by transfer RNA during translocation from the A site to the P site on the ribosome. J. Mol. Biol. 156, 113–140 (1982).
Fahnestock, S., Neumann, H. & Rich, A. Assay of ester and polyester formation by the ribosomal peptidyltransferase. Methods Enzymol. 30, 489–497 (1974).
Stern, S., Moazed, D. & Noller, H.F. Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Methods Enzymol. 164, 481–489 (1988).
We thank W. Wintermeyer for discussion and valuable comments on the manuscript, H.-J. Wieden for fMet-tRNAfMet(Flu) and Phe-tRNAPhe(QSY), Y.P. Semenkov and V.I. Katunin (Petersburg Nuclear Physics Institute) for generous gifts of tRNAs, D. Rodnin for ribosome preparations and A. Böhm, P. Striebeck, C. Schillings and S. Möbitz for expert technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft, the European Union, the Alfried Krupp von Bohlen und Halbach-Stiftung and the Fonds der Chemischen Industrie.
The authors declare no competing financial interests.
About this article
Cite this article
Bieling, P., Beringer, M., Adio, S. et al. Peptide bond formation does not involve acid-base catalysis by ribosomal residues. Nat Struct Mol Biol 13, 423–428 (2006). https://doi.org/10.1038/nsmb1091
Nature Structural & Molecular Biology (2014)
BMC Bioinformatics (2011)
The EMBO Journal (2011)