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.

1. Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.
2. Laboratory for Fluorescence Dynamics, Department of Physics, University of Illinois, Urbana-Champaign, Illinois 61801, USA.
3. Present addresses: National Institute of Child Health and Human Development, US National Institutes of Health, Bethesda, Maryland 20892, USA (Z.K.M.) and TransDerm Inc., 2161 Delaware Avenue, Santa Cruz, California 95060, USA (R.P.H).
4. These authors contributed equally to this work.

Correspondence to: Harry F Noller¹ e-mail: harry@nuvolari.ucsc.edu

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