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

Nature volume 468, pages 713–716 (2010)Cite this article

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Abstract

The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site1,2. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner3. Despite the availability of structures of various EF-G–ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G–ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit ‘pe/E’ hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the ‘missing link’ in terms of tRNA intermediates involved in the universally conserved translocation process.

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Figure 1: Substates I (TI PRE ) and II (TI POST ) of the 70S–EF-G–GDP–FA complex.
Figure 2: Localization and conformation of the tRNA of substates I (TIPRE) and II (TIPOST).
Figure 3: EF-G stabilizes the swivelled head movement in the TI POST state.
Figure 4: Model for translocation.

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Accession codes

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.

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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.

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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

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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 to Daniel 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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