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:

A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation

Nature Structural & Molecular Biology volume 15, pages 910–915 (2008)Cite this article

Abstract

EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the tRNAs on the ribosome, thereby reverting the canonical translocation reaction. In the current work, EF4 was directly visualized in the process of back-translocating tRNAs by single-particle cryo-EM. Using flexible fitting methods, we built a model of ribosome-bound EF4 based on the cryo-EM map and a recently published unbound EF4 X-ray structure. The cryo-EM map establishes EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which is present in a previously unseen, intermediate state and deviates markedly from the position of a canonical A-tRNA. Our results, therefore, provide insight into the underlying structural principles governing back-translocation.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: The cryo-EM reconstruction of the 70S–EF4 complex.
Figure 2: Molecular model of ribosome-bound EF4.
Figure 3: The A/L-tRNA.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Caldon, C.E., Yoong, P. & March, P.E. Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function. Mol. Microbiol. 41, 289–297 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Margus, T., Remm, M. & Tenson, T. Phylogenetic distribution of translational GTPases in bacteria. BMC Genomics 8, 15 (2007).

    Article  PubMed  Google Scholar 

  3. Qin, Y. et al. The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome. Cell 127, 721–733 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Colca, J.R. et al. Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics. J. Biol. Chem. 278, 21972–21979 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Wilson, D.N. & Nierhaus, K. The weird and wonderful world of bacterial ribosome regulation. Crit. Rev. Biochem. Mol. Biol. 42, 187–219 (2007).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Konevega, A.L. et al. Spontaneous reverse movement of mRNA-bound tRNA through the ribosome. Nat. Struct. Mol. Biol. 14, 318–324 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Frank, J. Three-Dimensional Electron Microscopy of Macromolecular Assemblies (Academic Press, New York, 1996).

    Google Scholar 

  9. Stark, H. et al. Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy. Cell 88, 19–28 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. 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  PubMed  Google Scholar 

  11. Berk, V., Zhang, W., Pai, R. & Cate, J. Structural basis for mRNA and tRNA positioning on the ribosome. Proc. Natl. Acad. Sci. USA 103, 15830–15834 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Evans, R.N., Blaha, G., Bailey, S. & Steitz, T. The structure of LepA, the ribosomal back translocase. Proc. Natl. Acad. Sci. USA 105, 4673–4678 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  15. Wriggers, W., Milligan, R.A. & McCammon, J.A. Situs: a package for docking crystal structures into low-resolution maps from electron microscopy. J. Struct. Biol. 125, 185–195 (1999).

    Article  CAS  PubMed  Google Scholar 

  16. Sali, A. & Blundell, T. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    Article  CAS  PubMed  Google Scholar 

  17. Connell, S. et al. Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors. Mol. Cell 25, 751–764 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Laurberg, M. et al. Structure of a mutant EF-G reveals domain III and possibly the fusidic acid binding site. J. Mol. Biol. 303, 593–603 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. Topf, M., Baker, M., John, B., Chiu, W. & Sali, A. Structural characterization of components of protein assemblies by comparative modeling and electron cryo-microscopy. J. Struct. Biol. 149, 191–203 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Topf, M. et al. Protein structure fitting and refinement guided by cryoEM density. Structure 16, 295–307 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  23. Nierhaus, K.H. Protein synthesis. An elongation factor turn-on. Nature 379, 491–492 (1996).

    Article  CAS  PubMed  Google Scholar 

  24. Wilson, K.S. & Noller, H.F. Molecular movement inside the translational engine. Cell 92, 337–349 (1998).

    Article  CAS  PubMed  Google Scholar 

  25. Savelsbergh, A. et al. An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation. Mol. Cell 11, 1517–1523 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Ogle, J.M. et al. Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292, 897–902 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Valle, M. et al. Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy. Nat. Struct. Biol. 10, 899–906 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Sanbonmatsu, K.Y., Joseph, S. & Tung, C. Simulating movement of tRNA into the ribosome during decoding. Proc. Natl. Acad. Sci. USA 102, 15854–15859 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Leach, K.L. et al. The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria. Mol. Cell 26, 393–402 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Nyborg, J. et al. Structure of the ternary complex of EF-Tu: macromolecular mimicry in translation. Trends Biochem. Sci. 21, 81–82 (1996).

    Article  CAS  PubMed  Google Scholar 

  31. Marquez, V., Wilson, D.N., Tate, W.P., Triana-Alonso, F. & Nierhaus, K.H. Maintaining the ribosomal reading frame: the influence of the E site during translational regulation of release factor 2. Cell 118, 45–55 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

    Article  CAS  PubMed  Google Scholar 

  33. Penczek, P.A., Frank, J. & Spahn, C. A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation. J. Struct. Biol. 154, 184–194 (2006).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M. Pech for his help and discussions, and A. Sali and W. Chiu for their support in the development of the cryo-EM density fitting methods. The present work was supported by grants from the Volkswagen Stiftung (C.M.T.S.), the Deutsche Forschungsgemeinschaft (DFG; SF8740 and SP 1130/2-1 to C.M.T.S. and K.H.N.), by the European Union 3D-EM Network of Excellence and by the European Union and Senatsverwaltung für Wissenschaft, Forschung und Kultur Berlin (UltraStructureNetwork, Anwenderzentrum). S.R.C. was supported with a grant from the Alexander von Humboldt Stiftung. M.T. is supported by a Medical Research Council Career Development Award (G0600084).

Author information

Author notes

  1. Yan Qin

    Present address: Present address: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.,

  2. Sean R Connell, Maya Topf and Yan Qin: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut für Medizinische Physik und Biophysik, Charite–Universitätsmedizin Berlin, Ziegelstrasse 5-9, Berlin, 10117, Germany

    Sean R Connell & Christian M T Spahn

  2. Cluster of Excellence for Macromolecular Complexes, Institut für Organische Chemie und Chemische Biologie, J.W. Goethe Universität, Max-von-Laue Strasse 7, Frankfurt am Main, 60438, Germany

    Sean R Connell & Paola Fucini

  3. Institute of Structural and Molecular Biology, School of Crystallography, Birkbeck College, Malet Street, London, WC1E 7HX, UK

    Maya Topf

  4. Max Planck Institute für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, Berlin, 14195, Germany

    Yan Qin, Daniel N Wilson, Paola Fucini & Knud H Nierhaus

  5. Gene Center and Department for Chemistry and Biochemistry, University of Munich, Feodor-Lynen-Strasse 25, Munich, 81377, Germany

    Daniel N Wilson

  6. Munich Center for Integrated Protein Science (CiPSM), University of Munich, Munich, 81377, Germany

    Daniel N Wilson

  7. UltraStrukturNetzwerk, Max Planck Institute für Molekulare Genetik, Ihnestrasse 73, 14195, Berlin, Germany

    Thorsten Mielke

Authors
  1. Sean R Connell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maya Topf
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yan Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel N Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thorsten Mielke
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Paola Fucini
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Knud H Nierhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christian M T Spahn
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christian M T Spahn.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–4, Supplementary Results and Supplementary Methods. (PDF 6212 kb)

Supplementary Movie

Resolution of the switch 1 region in the cryo-EM reconstruction. The switch 1 region of EF4 (red) has been modeled according to the corresponding region seen in the X-ray structure of EF-G-2 (PDB 1WDT). The cryo-EM density map is illustrated as a wire mesh and has been rendered at two thresholds. The switch 1 region (as modeled on the X-ray structure) does not directly correspond to the density and therefore it is presumed that it undergoes a conformational change on the ribosome. The cryo-EM density suggests that this conformational change would lead to the distal end of the loop forming an interaction near helix 14 (orange) of the 16S rRNA. The helical region in the distal end of the switch region appears to interact with domain III (of EF4; cyan) and H95 (purple) of the 23S rRNA. The movie was prepared with PyMOL. (MOV 4282 kb)

Supplementary Movie 2

A 360 degree rotation around the 3' CCA end of the A/L-tRNA (blue). The cryo-EM density is shown as a wire mesh and rendered at the relatively high 3 sigma threshold such that the various elements remain well defined although somewhat fragmented. The gap between the H71 and 92 (the H71/92 corridor) is clearly occupied by density, and it is within this density that the 3' CCA end of the A/L-tRNA has been modeled. (MOV 1675 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Connell, S., Topf, M., Qin, Y. et al. A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation. Nat Struct Mol Biol 15, 910–915 (2008). https://doi.org/10.1038/nsmb.1469

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1469

This article is cited by

Search

Advanced 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