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Dynamics of the context-specific translation arrest by chloramphenicol and linezolid

Nature Chemical Biology volume 16, pages 310–317 (2020)Cite this article

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Abstract

Chloramphenicol (CHL) and linezolid (LZD) are antibiotics that inhibit translation. Both were thought to block peptide-bond formation between all combinations of amino acids. Yet recently, a strong nascent peptide context-dependency of CHL- and LZD-induced translation arrest was discovered. Here we probed the mechanism of action of CHL and LZD by using single-molecule Förster resonance energy transfer spectroscopy to monitor translation arrest induced by antibiotics. The presence of CHL or LZD does not substantially alter dynamics of protein synthesis until the arrest-motif of the nascent peptide is generated. Inhibition of peptide-bond formation compels the fully accommodated A-site transfer RNA to undergo repeated rounds of dissociation and nonproductive rebinding. The glycyl amino-acid moiety on the A-site Gly-tRNA manages to overcome the arrest by CHL. Our results illuminate the mechanism of CHL and LZD action through their interactions with the ribosome, the nascent peptide and the incoming amino acid, perturbing elongation dynamics.

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Fig. 1: Context-specific inhibition of translation by CHL and LZD.
Fig. 2: Monitoring the drug-induced translation arrest using smFRET-based assay.
Fig. 3: Monitoring accommodation of A-site tRNA during CHL-induced translational arrest.
Fig. 4: Measuring FRET efficiency for the intermediate FRET state during CHL-induced translational arrest.
Fig. 5: The glycyl residue of the A-site tRNA rescues CHL-arrested ribosomes.
Fig. 6: Modeling of relative placement of antibiotics and tRNA substrates in the PTC active site.

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All experimental data are available upon reasonable request.

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All MATLAB scripts used in this study are available upon request.

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Acknowledgements

This work was supported by the US National Institutes of Health (grant nos. GM51266 to J.D.P. and R01 AI125518 to A.S.M. and N. V.-L.); by a Stanford Bio-X fellowship to J.C.; by the Knut and Alice Wallenberg Foundation postdoctoral scholarships to J.Z. and to J.W. (no. 2015.0406). We are grateful to H. Suga for providing the substrate for the flexizyme reaction and C. Innis for the advice and training in using the flexizyme-based procedures. We thank members of the Puglisi laboratory and the Mankin/Vázquez-Laslop laboratory for discussion and input.

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Author notes

  1. Junhong Choi

    Present address: Department of Genome Sciences, University of Washington, Seattle, WA, USA

  2. James Marks

    Present address: National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA

  3. These authors contributed equally: Junhong Choi, James Marks.

Authors and Affiliations

  1. Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA

    Junhong Choi, Jingji Zhang, Dong-Hua Chen, Jinfan Wang & Joseph D. Puglisi

  2. Department of Applied Physics, Stanford University, Stanford, CA, USA

    Junhong Choi

  3. Center for Biomolecular Sciences, University of Illinois, Chicago, IL, USA

    James Marks, Nora Vázquez-Laslop & Alexander S. Mankin

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Contributions

J.C., J.M., N.V.-L., A.S.M. and J.D.P. conceived the project and designed the single-molecule experiments. J.M. performed the toeprinting assay. J.C. prepared the reagent, and performed the single-molecule experiment and data analysis with the help of J.M. and J.W. J.Z. and D.-H.C. provided supporting data. J.C., J.M., J.Z., N.V.-L., A.S.M. and J.D.P. wrote the manuscript with the input of others.

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Correspondence to Nora Vázquez-Laslop, Alexander S. Mankin or Joseph D. Puglisi.

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Choi, J., Marks, J., Zhang, J. et al. Dynamics of the context-specific translation arrest by chloramphenicol and linezolid. Nat Chem Biol 16, 310–317 (2020). https://doi.org/10.1038/s41589-019-0423-2

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