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Allosteric control of the ribosome by small-molecule antibiotics

Nature Structural & Molecular Biology volume 19, pages 957–963 (2012)Cite this article

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Abstract

Protein synthesis is targeted by numerous, chemically distinct antibiotics that bind and inhibit key functional centers of the ribosome. Using single-molecule imaging and X-ray crystallography, we show that the aminoglycoside neomycin blocks aminoacyl–transfer RNA (aa-tRNA) selection and translocation as well as ribosome recycling by binding to helix 69 (H69) of 23S ribosomal RNA within the large subunit of the Escherichia coli ribosome. There, neomycin prevents the remodeling of intersubunit bridges that normally accompanies the process of subunit rotation to stabilize a partially rotated ribosome configuration in which peptidyl (P)-site tRNA is constrained in a previously unidentified hybrid position. Direct measurements show that this neomycin-stabilized intermediate is incompatible with the translation factor binding that is required for distinct protein synthesis reactions. These findings reveal the functional importance of reversible intersubunit rotation to the translation mechanism and shed new light on the allosteric control of ribosome functions by small-molecule antibiotics.

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Figure 1: Neomycin inhibits ribosome functions in vitro.
Figure 2: Neomycin stabilizes an intermediate conformation of the ribosome.
Figure 3: Neomycin contacts within H69 of 23S rRNA and bridging interactions with h45 of 16S rRNA induce global rearrangements in the 70S ribosome.
Figure 4: Position of tRNAPhe in the intermediate-rotated and neomycin-bound ribosome.
Figure 5: The intermediate ribosome configuration is incompatible with productive EF-G and EF-Tu binding.

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Acknowledgements

We thank R. Green (Johns Hopkins University) for providing the S13 knockout mouse strain, T. Suzuki (University of Tokyo) for providing the pKK3535 ribosome plasmids, K. Hamadani (University of California, Berkeley) for the RRF expression vector and M. O'Connor (University of Missouri-Kansas City) for helpful discussions throughout the course of this work. We also acknowledge helpful discussions and insights provided by all members of the Blanchard and Cate laboratories and J. Headd (Lawrence Berkeley National Laboratory) for help with PHENIX refinement. This work was supported by the US National Institutes of Health (2R01GM079238 to S.C.B., 1R01GM65050 to J.H.D.C. and National Cancer Institute grant CA92584 for the SIBYLS and 8.3.1 beamlines at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory), the Human Frontiers in Science Program (RGY0088), the National Science Foundation (0644129) and the US Department of Energy (DE-AC0376SF00098 for the SIBYLS and 8.3.1 beamlines at the ALS, Lawrence Berkeley National Laboratory). M.B.F. is a trainee in the Weill Cornell/Rockefeller University/Sloan-Kettering Tri-Institutional MD/PhD Program supported by US National Institutes of Health Medical Scientist Training Program grant GM07739. M.R.W. is supported by US National Institutes of Health National Research Service Award fellowship 5F31DC012026-02.

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  1. Leyi Wang, Arto Pulk, Michael R Wasserman and Michael B Feldman: These authors contributed equally to this work (L.W. and A.P., and M.R.W. and M.B.F.).

Authors and Affiliations

  1. Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA

    Leyi Wang, Michael R Wasserman, Roger B Altman & Scott C Blanchard

  2. Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA

    Arto Pulk & Jamie H Doudna Cate

  3. Department of Chemistry, University of California at Berkeley, Berkeley, California, USA

    Arto Pulk & Jamie H Doudna Cate

  4. Weill Cornell Medical College, Rockefeller University, Memorial Sloan-Kettering Cancer Center Tri-Institutional MD/PhD Program, New York, New York, USA

    Michael B Feldman

  5. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Jamie H Doudna Cate

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Contributions

L.W. and M.R.W. prepared dye-labeled ribosomes, tRNAs and translation factors and performed the smFRET imaging and analyzed the results. A.P. crystallized, collected, processed and refined X-ray data. L.W. and M.B.F. performed and analyzed bulk functional assays. L.W. and A.P. made the figures. R.B.A. prepared reagents crucial for smFRET experiments. S.C.B. and J.H.D.C. designed the study. All authors discussed the results and contributed to the writing of the manuscript.

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Correspondence to Jamie H Doudna Cate or Scott C Blanchard.

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Wang, L., Pulk, A., Wasserman, M. et al. Allosteric control of the ribosome by small-molecule antibiotics. Nat Struct Mol Biol 19, 957–963 (2012). https://doi.org/10.1038/nsmb.2360

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