Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites (original) (raw)

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Primary accessions

EMBL/GenBank/DDBJ

Protein Data Bank

Data deposits

The electron density maps and models of the TIPRE and the TIPOST complexes have been deposited in the 3D-EM database with accession numbers EMD-1798 and EMD-1799, and in the Protein Data Bank database with PDB IDs 2xsy, 2xtg, 2xux and 2xuy.

References

  1. Frank, J. & Spahn, C. M. The ribosome and the mechanism of protein synthesis. Rep. Prog. Phys. 69, 1383–1417 (2006)
    Article ADS CAS Google Scholar
  2. Schmeing, T. M. & Ramakrishnan, V. What recent ribosome structures have revealed about the mechanism of translation. Nature 461, 1234–1242 (2009)
    Article ADS CAS Google Scholar
  3. Shoji, S., Walker, S. E. & Fredrick, K. Ribosomal translocation: one step closer to the molecular mechanism. ACS Chem. Biol. 4, 93–107 (2009)
    Article CAS Google Scholar
  4. Ramakrishnan, V. Ribosome structure and the mechanism of translation. Cell 108, 557–572 (2002)
    Article 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. 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
  7. 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
  8. Munro, J. B., Sanbonmatsu, K. Y., Spahn, C. M. & Blanchard, S. C. Navigating the ribosome’s metastable energy landscape. Trends Biochem. Sci. 34, 390–400 (2009)
    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. Fischer, N. et al. Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy. Nature 466, 329–333 (2010)
    Article ADS CAS Google Scholar
  12. Valle, M. et al. Locking and unlocking of ribosomal motions. Cell 114, 123–134 (2003)
    Article CAS Google Scholar
  13. 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
  14. Connell, S. R. 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 Google Scholar
  15. Spahn, C. M. et al. Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO J. 23, 1008–1019 (2004)
    Article CAS Google Scholar
  16. Zhang, W., Dunkle, J. A. & Cate, J. H. Structures of the ribosome in intermediate states of ratcheting. Science 325, 1014–1017 (2009)
    Article ADS CAS Google Scholar
  17. Rodnina, M., 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
  18. 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
  19. Gao, Y. G. et al. The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 326, 694–699 (2009)
    Article ADS CAS Google Scholar
  20. Penczek, P. A., Frank, J. & Spahn, C. M. 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 Google Scholar
  21. Agrawal, R. K. et al. EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome. Nature Struct. Biol. 6, 643–647 (1999)
    Article CAS Google Scholar
  22. Scheres, S. H. et al. Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nature Methods 4, 27–29 (2007)
    Article CAS Google Scholar
  23. Matassova, A. B., Rodnina, M. V. & Wintermeyer, W. Elongation factor G-induced structural change in helix 34 of 16S rRNA related to translocation on the ribosome. RNA 7, 1879–1885 (2001)
    CAS PubMed PubMed Central Google Scholar
  24. Ticu, C. et al. Conformational changes in switch I of EF-G drive its directional cycling on and off the ribosome. EMBO J. 28, 2053–2065 (2009)
    Article CAS Google Scholar
  25. Spirin, A. S. The ribosome as a conveying thermal ratchet machine. J. Biol. Chem. 284, 21103–21119 (2009)
    Article CAS Google Scholar
  26. Schuette, J. C. et al. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J. 28, 755–765 (2009)
    Article CAS Google Scholar
  27. Whitford, P. C. et al. Accommodation of aminoacyl-tRNA into the ribosome involves reversible excursions along multiple pathways. RNA 16, 1196–1204 (2010)
    Article CAS Google Scholar
  28. Sharma, M. R. et al. Interaction of Era with the 30S ribosomal subunit implications for 30S subunit assembly. Mol. Cell 18, 319–329 (2005)
    Article CAS Google Scholar
  29. 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
  30. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)
    Article Google Scholar
  31. Chen, J. Z. & Grigorieff, N. SIGNATURE: a single-particle selection system for molecular electron microscopy. J. Struct. Biol. 157, 168–173 (2006)
    Article 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 Google Scholar
  33. Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006)
    Article ADS CAS Google Scholar
  34. Spahn, C. M. & Penczek, P. A. Exploring conformational modes of macromolecular assemblies by multiparticle cryo-EM. Curr. Opin. Struct. Biol. 19, 623–631 (2009)
    Article CAS Google Scholar
  35. Connell, S. R. et al. A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation. Nature Struct. Mol. Biol. 15, 910–915 (2008)
    Article CAS Google Scholar
  36. Whitford, P. C. et al. An all-atom structure-based potential for proteins: bridging minimal models with all-atom empirical forcefields. Proteins 75, 430–441 (2009)
    Article CAS Google Scholar
  37. Whitford, P. C. et al. Nonlocal helix formation is key to understanding S-adenosylmethionine-1 riboswitch function. Biophys. J. 96, L7–L9 (2009)
    Article Google Scholar
  38. Orzechowski, M. & Tama, F. Flexible fitting of high-resolution x-ray structures into cryoelectron microscopy maps using biased molecular dynamics simulations. Biophys. J. 95, 5692–5705 (2008)
    Article ADS CAS Google Scholar
  39. Lindahl, E., Hess, B. & van der Spoel, D. J. GROMACS 3.0: a package for molecular simulation and trajectory analysis. J. Mol. Model. 7, 306–317 (2001)
    Article CAS Google Scholar
  40. Berendsen, H. J. C., van der Spoel, D. & van Drunen, R. GROMACS: a message-passing parallel molecular dynamics implementation. Comput. Phys. Commun. 91, 43–56 (1995)
    Article ADS CAS Google Scholar
  41. Bocharov, E. V. et al. From structure and dynamics of protein L7/L12 to molecular switching in ribosome. J. Biol. Chem. 279, 17697–17706 (2004)
    Article CAS Google Scholar
  42. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996)
    Article CAS Google Scholar

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Acknowledgements

The present work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; SFB 740 TP A3 and TP Z1, SP 1130/2-1 to C.M.T.S., FU579 1-3 to P.F., HA 1672/7-5 to R.K.H. and WI3285/1-1 to D.N.W.), the European Union 3D-EM Network of Excellence (to C.M.T.S.), the European Union and Senatsverwaltung für Wissenschaft, Forschung und Kultur Berlin (UltraStructureNetwork, Anwenderzentrum) and US National Institutes of Health (NIH; grant GM 60635 to P.A.P.), the Cluster of Excellence ‘Macromolecular complexes’ at the Goethe University Frankfurt (DFG Project EXC 115 to P.F. and S.C.), and the Human Frontiers of Science Program Young Investigators Award HFSP67/07 (to P.F.). We thank the New Mexico Computing Application Center for generous time on the Encanto Supercomputer. P.C.W. is currently funded by a LANL Director’s Fellowship. This work was also supported by the Center for Theoretical Biological Physics sponsored by the National Science Foundation (NSF; grant PHY-0822283) with additional support from NSF-MCB-0543906, the LANL LDRD program and NIH grant R01-GM072686.

Author information

Authors and Affiliations

  1. Institut für Medizinische Physik und Biophysik, Charite – Universitätsmedizin Berlin, Ziegelstrasse 5–9, 10117-Berlin, Germany ,
    Andreas H. Ratje, Justus Loerke, Matthias Brünner, Peter W. Hildebrand, Thorsten Mielke & Christian M. T. Spahn
  2. Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, 35037, Germany
    Andreas H. Ratje & Roland K. Hartmann
  3. Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität, Feodor-Lynenstrasse 25, 81377 München, Germany,
    Aleksandra Mikolajka, Agata L. Starosta, Alexandra Dönhöfer & Daniel N. Wilson
  4. Center for Integrated Protein Science, Ludwig-Maximilians-Universität München, München, 81377, Germany
    Aleksandra Mikolajka & Daniel N. Wilson
  5. Frankfurt Institute for Molecular Life Sciences, Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Max-von Laue-Strasse 7, D-60438 Frankfurt am Main, Germany ,
    Sean R. Connell & Paola Fucini
  6. UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, 14195, Germany
    Thorsten Mielke
  7. Theoretical Division, Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, 87545, New Mexico, USA
    Paul C. Whitford & Karissa Y. Sanbonmatsu
  8. Center for Theoretical Biological Physics and Department of Physics, University of California, San Diego, La Jolla, 92093, California, USA
    José N. Onuchic
  9. Department of Computer Science, Florida State University, Tallahassee, 32306, Florida, USA
    Yanan Yu
  10. The University of Texas – Houston Medical School, 6431 Fannin, Houston, 77030, Texas, USA
    Pawel A. Penczek

Authors

  1. Andreas H. Ratje
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  2. Justus Loerke
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  3. Aleksandra Mikolajka
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  4. Matthias Brünner
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  5. Peter W. Hildebrand
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  6. Agata L. Starosta
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  7. Alexandra Dönhöfer
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  8. Sean R. Connell
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  9. Paola Fucini
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  10. Thorsten Mielke
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  11. Paul C. Whitford
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  12. José N. Onuchic
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  13. Yanan Yu
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  14. Karissa Y. Sanbonmatsu
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  15. Roland K. Hartmann
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  16. Pawel A. Penczek
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  17. Daniel N. Wilson
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  18. Christian M. T. Spahn
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Contributions

A.M., A.L.S. and A.D. prepared the complexes. A.H.R. and T.M. collected the cryoelectron microscopy data. A.H.R, J.L., M.B., S.R.C. and C.M.T.S. did the image processing. P.C.W., Y.Y., J.O. and K.Y.S. developed and employed the MDFit method. P.W.H. participated in docking and analysed the FA-binding site. A.H.R., R.K.H., S.R.C., P.F., P.A.P., D.N.W. and C.M.T.S. discussed the results and wrote the paper.

Corresponding authors

Correspondence toDaniel N. Wilson or Christian M. T. Spahn.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Results and Discussion, Supplementary Table 1, Supplementary Figures 1-7 with legends and Supplementary References. (PDF 1041 kb)

Supplementary Movie 1

The animation compares the cryo-EM maps of sub-state I (TIPRE) and sub-state II (TIPOST) of the 70S•EF-G•GDP•FA complex, which are shown as mesh superposed with docked molecular models in ribbons representation (see also legend to Fig. 1): EF-G (red), the tRNA (green), the 16S rRNA (yellow), and the 30S ribosomal proteins (pink). The maps are shown from the 50S side with the 50S subunit computationally removed. Alignment was based on the respective 50S subunits. (GIF 2823 kb)

Supplementary Movie 2

The animation compares the cryo-EM maps of sub-state I (TIPRE) and sub-state II (TIPOST) of the 70S•EF-G•GDP•FA complex, which are shown as mesh superposed with docked molecular models in ribbons representation (see also legend to Fig. 1): EF-G (red), the tRNA (green), the 16S rRNA (yellow), and the 30S ribosomal proteins (pink). The maps are shown as a top view onto the 30S subunit with the 50S subunit computationally removed. Alignment was based on the respective 50S subunits. (GIF 1272 kb)

Supplementary Movie 3

The animation compares the molecular models of sub-state I (TIPRE) and sub-state II (TIPOST) of the 70S•EF-G•GDP•FA complex in a close-up of the tRNA binding. The 30S subunit is shown with yellow ribbons, the tRNAs as blue ribbons and ribosomal residues that contact tRNAs in A-, P- and E-sites as spheres coloured magenta, green and orange, respectively (see also Fig. 2a, b). (GIF 1415 kb)

Supplementary Movie 4

In a common 50S alignment, the P/E-tRNA (green ribbon) of TIPRE (sub-state I) and the pe/EtRNA (magenta ribbon) of TIPOST (sub-state II) together with their respective mRNA codons11are compared to the positions of the tRNAs in classical A-, P- and E-sites and the mRNA (grey ribbons). See also Fig. 3d. (GIF 145 kb)

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Ratje, A., Loerke, J., Mikolajka, A. et al. Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites.Nature 468, 713–716 (2010). https://doi.org/10.1038/nature09547

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