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Peptide bond formation does not involve acid-base catalysis by ribosomal residues

Abstract

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.

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Figure 1: Chemistry of peptide bond formation.
Figure 2: Kinetics of accommodation and PT reaction with Phe-tRNAPhe as substrate.
Figure 3: Correct positioning of Phelac-tRNAPhe at the PT center.
Figure 4: Kinetics of fMet-Phelac formation.
Figure 5: Summary of rate constants.

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Acknowledgements

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.

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Correspondence to Marina V Rodnina.

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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

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