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:

Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy

Abstract

The translocation step of protein synthesis entails large-scale rearrangements of the ribosome–transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during translocation by time-resolved single-particle electron cryomicroscopy (cryo-EM). Unbiased computational sorting of cryo-EM images yielded 50 distinct three-dimensional reconstructions, showing the tRNAs in classical, hybrid and various novel intermediate states that provide trajectories and kinetic information about tRNA movement through the ribosome. The structures indicate how tRNA movement is coupled with global and local conformational changes of the ribosome, in particular of the head and body of the small ribosomal subunit, and show that dynamic interactions between tRNAs and ribosomal residues confine the path of the tRNAs through the ribosome. The temperature dependence of ribosome dynamics reveals a surprisingly flat energy landscape of conformational variations at physiological temperature. The ribosome functions as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to directed movement.

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: Trajectories of tRNA movement during (retro-)translocation.
Figure 2: Dynamics of ribosome–tRNA interactions and of the 30S subunit.
Figure 3: Positions of the E-site tRNA.
Figure 4: Coupling between tRNA movement and 30S dynamics.
Figure 5: Increase of ribosome dynamics with temperature.

Similar content being viewed by others

Accession codes

Data deposits

The three-dimensional density maps have been deposited in the Electron Microscopy Data Bank at the European Biology Laboratory—European Bioinformatics Institute—under accession codes EMD-1716 to EMD-1720 (pre1–5), EMD-1721 to EMD-1723 (post1–3), EMD-1724 (sub-state of post-translocation complex at 18 °C), and EMD-1725 to EMD-1727 (post-translocation complex at 18 °C, 4 °C and 37 °C), respectively.

References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Gavrilova, L. P. et al. Factor-free (“non-enzymic”) and factor-dependent systems of translation of polyuridylic acid by Escherichia coli ribosomes. J. Mol. Biol. 101, 537–552 (1976)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  6. Cornish, P. V., Ermolenko, D. N., Noller, H. F. & Ha, T. Spontaneous intersubunit rotation in single ribosomes. Mol. Cell 30, 578–588 (2008)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Agirrezabala, X. et al. Visualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome. Mol. Cell 32, 190–197 (2008)

    Article  CAS  Google Scholar 

  10. Julian, P. et al. Structure of ratcheted ribosomes with tRNAs in hybrid states. Proc. Natl Acad. Sci. USA 105, 16924–16927 (2008)

    Article  ADS  CAS  Google Scholar 

  11. Marshall, R. A., Dorywalska, M. & Puglisi, J. D. Irreversible chemical steps control intersubunit dynamics during translation. Proc. Natl Acad. Sci. USA 105, 15364–15369 (2008)

    Article  ADS  CAS  Google Scholar 

  12. Blanchard, S. C. et al. tRNA dynamics on the ribosome during translation. Proc. Natl Acad. Sci. USA 101, 12893–12898 (2004)

    Article  ADS  CAS  Google Scholar 

  13. Kim, H. D., Puglisi, J. D. & Chu, S. Fluctuations of transfer RNAs between classical and hybrid states. Biophys. J. 93, 3575–3582 (2007)

    Article  ADS  CAS  Google Scholar 

  14. Munro, J. B., Altman, R. B., O’Connor, N. & Blanchard, S. C. Identification of two distinct hybrid state intermediates on the ribosome. Mol. Cell 25, 505–517 (2007)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  17. 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 

  18. Jenner, L., Rees, B., Yusupov, M. & Yusupova, G. Messenger RNA conformations in the ribosomal E site revealed by X-ray crystallography. EMBO Rep. 8, 846–850 (2007)

    Article  CAS  Google Scholar 

  19. Rodnina, M. V., Savelsbergh, A., Katunin, V. I. & Wintermeyer, W. Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome. Nature 385, 37–41 (1997)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Savelsbergh, A. et al. Control of phosphate release from elongation factor G by ribosomal protein L7/12. EMBO J. 24, 4316–4323 (2005)

    Article  CAS  Google Scholar 

  22. 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 

  23. Cornish, P. V. et al. Following movement of the L1 stalk between three functional states in single ribosomes. Proc. Natl Acad. Sci. USA 106, 2571–2576 (2009)

    Article  ADS  CAS  Google Scholar 

  24. Fei, J., Kosuri, P., MacDougall, D. D. & Gonzalez, R. L. Coupling of ribosomal L1 stalk and tRNA dynamics during translation elongation. Mol. Cell 30, 348–359 (2008)

    Article  CAS  Google Scholar 

  25. Fei, J. et al. Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation. Proc. Natl Acad. Sci. USA 106, 15702–15707 (2009)

    Article  ADS  CAS  Google Scholar 

  26. Munro, J. B. et al. Spontaneous formation of the unlocked state of the ribosome is a multistep process. Proc. Natl Acad. Sci. USA 107, 709–714 (2010)

    Article  ADS  CAS  Google Scholar 

  27. Schuwirth, B. S. et al. Structures of the bacterial ribosome at 3.5 Å resolution. Science 310, 827–834 (2005)

    Article  ADS  CAS  Google Scholar 

  28. Villa, E. et al. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc. Natl Acad. Sci. USA 106, 1063–1068 (2009)

    Article  ADS  CAS  Google Scholar 

  29. Zhang, W., Dunkle, J. A. & Cate, J. H. D. Structures of the ribosome in intermediate states of ratcheting. Science 325, 1014–1017 (2009)

    Article  ADS  CAS  Google Scholar 

  30. Robertson, J. M., Paulsen, H. & Wintermeyer, W. Pre-steady-state kinetics of ribosomal translocation. J. Mol. Biol. 192, 351–360 (1986)

    Article  CAS  Google Scholar 

  31. Paulsen, H. & Wintermeyer, W. tRNA topography during translocation: steady-state and kinetic fluorescence energy-transfer studies. Biochemistry 25, 2749–2756 (1986)

    Article  CAS  Google Scholar 

  32. Cordova, N. J., Ermentrout, B. & Oster, G. F. Dynamics of single-motor molecules—the thermal ratchet model. Proc. Natl Acad. Sci. USA 89, 339–343 (1992)

    Article  ADS  CAS  Google Scholar 

  33. Astumian, R. D. Thermodynamics and kinetics of a Brownian motor. Science 276, 917–922 (1997)

    Article  CAS  Google Scholar 

  34. Ermolenko, D. N. et al. Observation of intersubunit movement of the ribosome in solution using FRET. J. Mol. Biol. 370, 530–540 (2007)

    Article  CAS  Google Scholar 

  35. Lancaster, L. E. et al. Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes. Mol. Microbiol. 69, 390–401 (2008)

    Article  CAS  Google Scholar 

  36. Taylor, D. J. et al. Structures of modified eEF2·80S ribosome complexes reveal the role of GTP hydrolysis in translocation. EMBO J. 26, 2421–2431 (2007)

    Article  CAS  Google Scholar 

  37. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228 (1988)

    Article  CAS  Google Scholar 

  38. Bellare, J. R., Davis, H. T., Scriven, L. E. & Talmon, Y. Controlled environment vitrification system—an improved sample preparation technique. J. Electron Microsc. Tech. 10, 87–111 (1988)

    Article  CAS  Google Scholar 

  39. Sander, B., Golas, M. M. & Stark, H. Advantages of CCD detectors for de novo three-dimensional structure determination in single-particle electron microscopy. J. Struct. Biol. 151, 92–105 (2005)

    Article  CAS  Google Scholar 

  40. Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: Semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)

    Article  CAS  Google Scholar 

  41. Sander, B., Golas, M. M. & Stark, H. Automatic CTF correction for single particles based upon multivariate statistical analysis of individual power spectra. J. Struct. Biol. 142, 392–401 (2003)

    Article  CAS  Google Scholar 

  42. Bhat, T. N. & Cohen, G. H. Omitmap—an electron-density map suitable for the examination of errors in a macromolecular model. J. Appl. Crystallogr. 17, 244–248 (1984)

    Article  CAS  Google Scholar 

  43. van Heel, M. et al. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996)

    Article  CAS  Google Scholar 

  44. Sander, B., Golas, M. M. & Stark, H. Corrim-based alignment for improved speed in single-particle image processing. J. Struct. Biol. 143, 219–228 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Blau for technical help in preparing Fig. 4. Research in the laboratory of H.S. was supported by grants from the Federal Ministry of Education and Research (BMBF), Germany, the Sixth Framework Programme of the European Union via the Integrated Project 3DRepertoire, by the state of Lower-Saxony and the Volkswagen Foundation, Hannover, Germany. N.F. was supported by a Boehringer-Ingelheim fellowship. M.V.R. and W.W. were supported by grants from the Deutsche Forschungsgemeinschaft.

Author information

Authors and Affiliations

Authors

Contributions

N.F. conceived and performed the cryo-EM experiments and data analysis with mentoring by H.S.; A.L.K. prepared complexes for the analysis; N.F., A.L.K., W.W., M.V.R. and H.S. discussed results and wrote the manuscript.

Corresponding author

Correspondence to Holger Stark.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures 1- 11 with legends, Supplementary Tables 1 - 4, References and full legends for Supplementary Movies 1- 6 and 3D PDFs. (PDF 3144 kb)

Supplementary Movie 1

GIF-animation visualizing ribosome dynamics and tRNA movement during retro-translocation as revealed by unsupervised 2D classification (see Supplementary Information file for full legend). (GIF 4451 kb)

Supplementary Movie 2

The movie shows a sequence of 20 ribosome 3D structures of sub-states along the (retro-) translocation pathway arranged according to the smallest free energy differences between subsequent sub-states (see Supplementary Information file for full legend). (MPG 1514 kb)

Supplementary Movie 3

GIF-animation of the same sequence of 20 sub-states as in Supplementary Movie 2 (see Supplementary Information file for full legend). (GIF 1847 kb)

Supplementary Movie 4

GIF-animation showing a sequence of 11 different sub-states of the classic pre-translocation state pre1 from top (upper row) and the solvent side of the 30S subunit (lower left) (see Supplementary Information file for full legend). (GIF 1407 kb)

Supplementary Movie 5

GIF-animation similar to Supplementary Movie 2, but depicting only 11 sub states (see Supplementary Information file for full legend). (GIF 1307 kb)

Supplementary Movie 6

Movie visualizing the temperature dependence of ribosome dynamics by interpolating between cryo-EM maps of the same post-translocation complex prepared at different temperatures (see Supplementary Information file for full legend). (MPG 6651 kb)

Supplementary 3D PDFs

Nine isosurface representations of sub-states of the 70S˙fMetVal tRNAVal˙tRNAfMet complex (see Supplementary Information file for full legend). (PDF 24002 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fischer, N., Konevega, A., Wintermeyer, W. et al. Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy. Nature 466, 329–333 (2010). https://doi.org/10.1038/nature09206

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09206

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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