Functional Dynamics within the Human Ribosome Regulate the Rate of Active Protein Synthesis

Angelica Ferguson, Leyi Wang, Roger B. Altman, Daniel S. Terry, Manuel F. Juette, Benjamin J. Burnett, Jose L. Alejo, Randall A. Dass, Matthew M. Parks, C. Theresa Vincent, Scott C. Blanchard

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation.

Original languageEnglish (US)
Pages (from-to)475-486
Number of pages12
JournalMolecular Cell
Volume60
Issue number3
DOIs
StatePublished - Nov 5 2015

Bibliographical note

Funding Information:
We are grateful to Drs. Christian Spahn, Tatyana Budkevich, and Kaori Yamamoto for purified elongation factors. We also thank the Weill Cornell Genomics and Advanced Bioinformatics Cores for contributions to processing of ribosome profiling data and the Rockefeller University Proteomics Resource Center for mass spectrometry. This work was supported by grants from the NIH (GM 079238) (to S.C.B.), the Tri-Institutional Stem Cell Initiative funded by the Starr Foundation (to S.C.B. and C.T.V.), the Tri-Institutional Training Program in Chemical Biology (to A.F.), the German Academic Exchange Service (to M.F.J.) as well as the Swedish Research Council and VINNMER–Marie Curie International Qualification (to C.T.V.) for collaborative initiatives between Weill Cornell and the Karolinska Institute.

Publisher Copyright:
© 2015 Elsevier Inc.

Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.

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