Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The antibiotic viomycin traps the ribosome in an intermediate state of translocation

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Positions of FRET pairs in the 70S ribosome.
Figure 2: Changes in FRET efficiency induced by viomycin.
Figure 3: Chemical footprinting of a ribosome–tRNAfMet–viomycin complex.
Figure 4: Chemical footprinting of a 70S ribosome–tRNAfMetN-Ac-Phe-tRNAPhe–viomycin complex.

Similar content being viewed by others

References

  1. Spirin, A.S. Ribosomal translocation: facts and models. Prog. Nucleic Acid Res. Mol. Biol. 32, 75–114 (1985).

    Article  CAS  Google Scholar 

  2. Belitsina, N.V., Glukhova, M.A. & Spirin, A.S. Translocation in ribosomes by attachment-detachment of elongation factor G without GTP cleavage: evidence from a column-bound ribosome system. FEBS Lett. 54, 35–38 (1975).

    Article  CAS  Google Scholar 

  3. Cukras, A.R., Southworth, D.R., Brunelle, J.L., Culver, G.M. & Green, R. Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex. Mol. Cell 12, 321–328 (2003).

    Article  CAS  Google Scholar 

  4. Fredrick, K. & Noller, H.F. Catalysis of ribosomal translocation by sparsomycin. Science 300, 1159–1162 (2003).

    Article  CAS  Google Scholar 

  5. Gavrilova, L.P. & Spirin, A.S. Stimulation of “non-enzymic” translocation in ribosomes by p-chloromercuribenzoate. FEBS Lett. 17, 324–326 (1971).

    Article  CAS  Google Scholar 

  6. Inoue-Yokosawa, N., Ishikawa, C. & Kaziro, Y. The role of guanosine triphosphate in translocation reaction catalyzed by elongation factor G. J. Biol. Chem. 249, 4321–4323 (1974).

    CAS  PubMed  Google Scholar 

  7. Pestka, S. Studies on the formation of transfer ribonucleic acid-ribosome complexes. 3. The formation of peptide bonds by ribosomes in the absence of supernatant enzymes. J. Biol. Chem. 243, 2810–2820 (1968).

    CAS  PubMed  Google Scholar 

  8. Pestka, S. Studies on the formation of transfer ribonucleic acid-ribosome complexes. VI. Oligopeptide synthesis and translocation on ribosomes in the presence and absence of soluble transfer factors. J. Biol. Chem. 244, 1533–1539 (1969).

    CAS  PubMed  Google Scholar 

  9. Korostelev, A., Trakhanov, S., Laurberg, M. & Noller, H.F. Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements. Cell 126, 1065–1077 (2006).

    Article  CAS  Google Scholar 

  10. Yusupov, M.M. et al. Crystal structure of the ribosome at 5.5 Å resolution. Science 292, 883–896 (2001).

    Article  CAS  Google Scholar 

  11. Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006).

    Article  CAS  Google Scholar 

  12. Agrawal, R.K. et al. Visualization of tRNA movements on the Escherichia coli 70S ribosome during the elongation cycle. J. Cell Biol. 150, 447–460 (2000).

    Article  CAS  Google Scholar 

  13. Valle, M. et al. Locking and unlocking of ribosomal motions. Cell 114, 123–134 (2003).

    Article  CAS  Google Scholar 

  14. Bretscher, M.S. Translocation in protein synthesis: a hybrid structure model. Nature 218, 675–677 (1968).

    Article  CAS  Google Scholar 

  15. Spirin, A.S. A model of the functioning ribosome: locking and unlocking of the ribosome subparticles. Cold Spring Harb. Symp. Quant. Biol. 34, 197–207 (1969).

    Article  CAS  Google Scholar 

  16. Moazed, D. & Noller, H.F. Intermediate states in the movement of transfer RNA in the ribosome. Nature 342, 142–148 (1989).

    Article  CAS  Google Scholar 

  17. Frank, J. & Agrawal, R.K. A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406, 318–322 (2000).

    Article  CAS  Google Scholar 

  18. Modolell, J. & Vazquez The inhibition of ribosomal translocation by viomycin. Eur. J. Biochem. 81, 491–497 (1977).

    Article  CAS  Google Scholar 

  19. Peske, F., Savelsbergh, A., Katunin, V.I., Rodnina, M.V. & Wintermeyer, W. Conformational changes of the small ribosomal subunit during elongation factor G-dependent tRNA-mRNA translocation. J. Mol. Biol. 343, 1183–1194 (2004).

    Article  CAS  Google Scholar 

  20. Johansen, S.K., Maus, C.E., Plikaytis, B.B. & Douthwaite, S. Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2′-O-methylations in 16S and 23S rRNAs. Mol. Cell 23, 173–182 (2006).

    Article  CAS  Google Scholar 

  21. Moazed, D. & Noller, H.F. Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping sites in the peptidyl transferase region of 23S ribosomal RNA. Biochimie 69, 879–884 (1987).

    Article  CAS  Google Scholar 

  22. Powers, T. & Noller, H.F. Selective perturbation of G530 of 16 S rRNA by translational miscoding agents and a streptomycin-dependence mutation in protein S12. J. Mol. Biol. 235, 156–172 (1994).

    Article  CAS  Google Scholar 

  23. Yamada, T., Mizugichi, Y., Nierhaus, K.H. & Wittmann, H.G. Resistance to viomycin conferred by RNA of either ribosomal subunit. Nature 275, 460–461 (1978).

    Article  CAS  Google Scholar 

  24. Yamada, T. & Bierhaus, K.H. Viomycin favours the formation of 70S ribosome couples. Mol. Gen. Genet. 161, 261–265 (1978).

    Article  CAS  Google Scholar 

  25. Pan, D., Kirillov, S.V. & Cooperman, B.S. Kinetically competent intermediates in the translocation step of protein synthesis. Mol. Cell 25, 519–529 (2007).

    Article  CAS  Google Scholar 

  26. Hickerson, R., Majumdar, Z.K., Baucom, A., Clegg, R.M. & Noller, H.F. Measurement of internal movements within the 30 S ribosomal subunit using Forster resonance energy transfer. J. Mol. Biol. 354, 459–472 (2005).

    Article  CAS  Google Scholar 

  27. Majumdar, Z.K., Hickerson, R., Noller, H.F. & Clegg, R.M. Measurements of internal distance changes of the 30S ribosome using FRET with multiple donor-acceptor pairs: quantitative spectroscopic methods. J. Mol. Biol. 351, 1123–1145 (2005).

    Article  CAS  Google Scholar 

  28. Lieberman, K.R. et al. The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit. J. Mol. Biol. 297, 1129–1143 (2000).

    Article  CAS  Google Scholar 

  29. Gao, H. et al. Study of the structural dynamics of the E. coli 70S ribosome using real-space refinement. Cell 113, 789–801 (2003).

    Article  CAS  Google Scholar 

  30. Moazed, D. & Noller, H.F. Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell 57, 585–597 (1989).

    Article  CAS  Google Scholar 

  31. Moazed, D. & Noller, H.F. Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA. J. Mol. Biol. 211, 135–145 (1990).

    Article  CAS  Google Scholar 

  32. Dorner, S., Brunelle, J.L., Sharma, D. & Green, R. The hybrid state of tRNA binding is an authentic translation elongation intermediate. Nat. Struct. Mol. Biol. 13, 234–241 (2006).

    Article  CAS  Google Scholar 

  33. Bycroft, B.W. The crystal structure of viomycin, a tuberculostatic antibiotic. JCS Chem. Commun. 660–661 (1972).

  34. Shoji, S., Walker, S.E. & Fredrick, K. Reverse translocation of tRNA in the ribosome. Mol. Cell 24, 931–942 (2006).

    Article  CAS  Google Scholar 

  35. Wilson, K.S. & Noller, H.F. Mapping the position of translational elongation factor EF-G in the ribosome by directed hydroxyl radical probing. Cell 92, 131–139 (1998).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Harry F Noller.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1243

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing