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Substrate-assisted catalysis of peptide bond formation by the ribosome

Nature Structural & Molecular Biology volume 11, pages 1101–1106 (2004)Cite this article

Abstract

The ribosome accelerates the rate of peptide bond formation by at least 107-fold, but the catalytic mechanism remains controversial. Here we report evidence that a functional group on one of the tRNA substrates plays an essential catalytic role in the reaction. Substitution of the P-site tRNA A76 2′ OH with 2′ H or 2′ F results in at least a 106-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. Such substrate-assisted catalysis is relatively uncommon among modern protein enzymes, but it is a property predicted to be essential for the evolution of enzymatic function. These results suggest that substrate assistance has been retained as a catalytic strategy during the evolution of the prebiotic peptidyl transferase center into the modern ribosome.

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Figure 1: The peptidyl transferase reaction and characterization of modified tRNALys containing A76, dA76 or fA76.
Figure 2: The rapid kinetic peptidyl transfer assay.
Figure 3: Peptidyl donor activity of modified tRNAs.
Figure 4: P-site occupancy by modified tRNAs.

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Acknowledgements

We thank A. Ambrogelly for the gift of E. coli lysyl-tRNA synthetase and A. Weiner for the gift of pET-22-CCA plasmid containing His6-tagged CCA-adding enzyme. We thank E. De La Cruz for helpful discussions and J. Lorsch and T. Steitz for comments on the manuscript. This work was supported by an American Cancer Society Beginning Investigator award to S.A.S., a US National Science Foundation predoctoral fellowship to J.S.W., an Erwin Schroedinger fellowship (J2172) to S.D. and US National Institutes of Health R01 and Howard Hughes Medical Institute funding for R.G.

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  1. Joshua S Weinger and K Mark Parnell: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, 06520-8114, Connecticut, USA

    Joshua S Weinger, K Mark Parnell & Scott A Strobel

  2. Department of Molecular Biology and Genetics, Johns Hopkins University Medical School, Howard Hughes Medical Institute, Baltimore, 21205, Maryland, USA

    Silke Dorner & Rachel Green

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Correspondence to Scott A Strobel.

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Weinger, J., Parnell, K., Dorner, S. et al. Substrate-assisted catalysis of peptide bond formation by the ribosome. Nat Struct Mol Biol 11, 1101–1106 (2004). https://doi.org/10.1038/nsmb841

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