Abstract
Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.
Original language | English (US) |
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Pages (from-to) | E8603-E8610 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 114 |
Issue number | 41 |
DOIs | |
State | Published - Oct 10 2017 |
Bibliographical note
Funding Information:ACKNOWLEDGMENTS. We thank R. Green (Johns Hopkins University) for providing the S13 knockout strain, P. Schultz (Scripps Research Institute) for providing the L5 knockout strain, and D. Wilson and P. Huter (Ludwig-Maximilian University Munich) for providing purified EF-P and its mutant counterpart. We also acknowledge helpful discussions, insights and co-operation provided by all members of the S.C.B. laboratory, especially Michael Wasserman, Roger Altman, Mikael Holm, and Randall Dass. This work was supported by the US NIH (Grant 5R01GM079238) and the National Science Foundation (Grant MCB-1412353).
Publisher Copyright:
© 2017, National Academy of Sciences. All rights reserved.
Keywords
- Aminoglycosides
- EF-P
- Fidelity
- Ribosome
- Translocation