Abstract
During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.
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Acknowledgements
These studies were supported by US National Institutes of Health grant GM-17129 and US National Science Foundation grant MCB-0212689 (to H.F.N.), US National Institutes of Health grant PHS-5P41RR03155 (to R.M.C.), a NATO-NSF postdoctoral fellowship (to D.N.E.), a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund (to P.C.S.) and an Else Adler Postdoctoral Fellowship from the Damon Runyon Cancer Research Foundation and a postdoctoral fellowship from the Ford Foundation (to R.P.H). We thank members of the Noller laboratory for helpful discussions.
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D.N.E., P.C.S., R.P.H. and H.F.N. designed the experiments; D.N.E., Z.K.M. and R.P.H. performed the FRET experiments; D.N.E., Z.K.M. and R.M.C. analyzed the FRET data, P.C.S. performed the chemical probing experiments, D.N.E., P.C.S. and H.F.N. wrote the manuscript and all authors contributed to the final version of the manuscript.
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Supplementary information
Supplementary Fig. 1
Chemical footprinting of a 70S ribosome–tRNAPhe–viomycin complex. (PDF 151 kb)
Supplementary Table 1
Translocation activities of reconstituted, fluorescently-labeled ribosomes. (PDF 42 kb)
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Ermolenko, D., Spiegel, P., Majumdar, Z. et al. The antibiotic viomycin traps the ribosome in an intermediate state of translocation. Nat Struct Mol Biol 14, 493–497 (2007). https://doi.org/10.1038/nsmb1243
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DOI: https://doi.org/10.1038/nsmb1243
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