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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.

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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.

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)

    CAS  Article  Google Scholar 

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

    CAS  Article  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)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  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)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  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)

    CAS  Article  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)

    ADS  CAS  Article  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)

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  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)

    ADS  CAS  Article  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)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    ADS  CAS  Article  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)

    CAS  Article  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)

    ADS  CAS  Article  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)

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  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)

    ADS  CAS  Article  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)

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  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)

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  Google Scholar 

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

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  Google Scholar 

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

    CAS  Article  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)

    CAS  Article  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.

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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)

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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

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