A two-step chemical mechanism for ribosome-catalysed peptide bond formation

Abstract

The chemical step of natural protein synthesis, peptide bond formation, is catalysed by the large subunit of the ribosome. Crystal structures have shown that the active site for peptide bond formation is composed entirely of RNA1. Recent work has focused on how an RNA active site is able to catalyse this fundamental biological reaction at a suitable rate for protein synthesis. On the basis of the absence of important ribosomal functional groups2 , lack of a dependence on pH3, and the dominant contribution of entropy to catalysis4, it has been suggested that the role of the ribosome is limited to bringing the substrates into close proximity. Alternatively, the importance of the 2′-hydroxyl of the peptidyl-transfer RNA5 and a Brønsted coefficient near zero6 have been taken as evidence that the ribosome coordinates a proton-transfer network. Here we report the transition state of peptide bond formation, based on analysis of the kinetic isotope effect at five positions within the reaction centre of a peptidyl-transfer RNA mimic. Our results indicate that in contrast to the uncatalysed reaction, formation of the tetrahedral intermediate and proton transfer from the nucleophilic nitrogen both occur in the rate-limiting step. Unlike in previous proposals, the reaction is not fully concerted; instead, breakdown of the tetrahedral intermediate occurs in a separate fast step. This suggests that in addition to substrate positioning, the ribosome is contributing to chemical catalysis by changing the rate-limiting transition state.

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Figure 1: Proposed reaction mechanisms.
Figure 2: Histograms of measured isotope effects.
Figure 3: Kinetic isotope effects.
Figure 4: Structures of the substrates and transition states for peptide bond formation shown in similar orientations.

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Acknowledgements

We thank J. Klinman and D. Singleton for critical reading of the manuscript, and members of the S.A.S. laboratory for discussion. This work was supported by an NIH postdoctoral fellowship (D.A.H.), a Brown-Coxe fellowship (V.S.) and an NIH grant (GM54839).

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D.A.H. and S.A.S. devised experiments, M.Z. synthesized substrates, D.A.H. collected and analysed isotope effect data, V.S. calculated theoretical isotope effects, and D.A.H. and S.A.S. wrote the paper.

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Correspondence to Scott A. Strobel.

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The authors declare no competing financial interests.

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Hiller, D., Singh, V., Zhong, M. et al. A two-step chemical mechanism for ribosome-catalysed peptide bond formation. Nature 476, 236–239 (2011). https://doi.org/10.1038/nature10248

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