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Structure of eEF3 and the mechanism of transfer RNA release from the E-site

Nature volume 443, pages 663–668 (2006)Cite this article

Abstract

Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA–eEF1A–GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.

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Figure 1: Structures of Saccharomyces cerevisiae eEF3 and nucleotide binding.
Figure 2: Reconstitution and cryo-EM structure of the eEF3–ribosome complex.
Figure 3: Dynamic behaviour of eEF3 and the 80S ribosome.
Figure 4: Model of the role of eEF3 in the fungal elongation cycle.

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Acknowledgements

G.R.A. was supported by the NIH, HFSP, EU FP5 and SNF. T.G.K. was supported by the NIH and HFSP. We are grateful to I. Kerr and D. Brodersen for discussions, and to L. Van for technical assistance. The work was further supported by grants from the VolkswagenStiftung (to R.B. and C.M.T.S.), the Deutsche Forschungsgemeinschaft (to R.B.), and by the European Union and Senatsverwaltung für Wissenschaft, Forschung und Kultur Berlin (UltraStructureNetwork).

Author information

Author notes

  1. Thomas Becker, Mario Halic & Roland Beckmann

    Present address: Gene Center and Department of Chemistry & Biochemistry, University of Munich, Feodor-Lynen-Strasse 25, 81377, Munich, Germany

  2. Christian B. F. Andersen and Thomas Becker: *These authors contributed equally to this work

Authors and Affiliations

  1. Centre for Structural Biology, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000, Aarhus, Denmark

    Christian B. F. Andersen, Thomas Boesen & Gregers R. Andersen

  2. Institute of Biochemistry, Charité, University Medical School, Humboldt University of Berlin, Monbijoustrasse 2, 10117, Berlin, Germany

    Thomas Becker, Michael Blau, Mario Halic & Roland Beckmann

  3. Department of Molecular Genetics, Microbiology & Immunology, UMDNJ Robert Wood Johnson Medical School, Piscataway, New Jersey, 08854-5635, USA

    Monika Anand, Bharvi Balar & Terri Goss Kinzy

  4. Immunology, UMDNJ Robert Wood Johnson Medical School,

    Monika Anand, Bharvi Balar & Terri Goss Kinzy

  5. UltraStructureNetwork, USN, Max Planck Institute for Molecular Genetics, Ihnestrasse 63-73, 14195, Berlin, Germany

    Thorsten Mielke

  6. Department of Chemistry and iNANO Interdisciplinary Nanoscience Center, University of Aarhus, Langelandsgade 140, DK-8000, Aarhus, Denmark

    Jan Skov Pedersen

  7. Institut für Medizinische Physik und Biophysik, Charité, University Medical School, Humboldt University of Berlin, Ziegelstrasse 5-9, 10117, Berlin, Germany

    Christian M. T. Spahn

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Corresponding authors

Correspondence to Gregers R. Andersen or Roland Beckmann.

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Competing interests

Coordinates and structure factors for the crystal structures have been deposited in the RCSB Protein Data Bank under accession numbers 2IX3, 2IW3 and 2IWH; the electron microscopy (EM) map has been deposited in the EMBL-European Bioinformatics Institute EM Data Bank under accession number EMD-1233 and coordinates of EM-based models in the RCSB Protein Data Bank under accession number 2IX8. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Sequence of Saccharomyces cerevisiae eEF3. (PDF 40 kb)

Supplementary Figure 2

a, Stereo view of ADPNP bound to eEF3 at the unusual nucleotide binding site. b, The unusual nucleotide binding site of eEF3 compared to the canonical site modelled form the structure of RLI. c, The sulphate binding site formed by the signature motif (shown in green). (PDF 714 kb)

Supplementary Figure 3

Multiple alignment of eEF3 sequences. (PDF 199 kb)

Supplementary Figure 4

SAXS analysis of eEF3. (PDF 447 kb)

Supplementary Figure 5

Nucleotide dependency of eEF3 binding. (PDF 188 kb)

Supplementary Figure 6

Complete eEF3-RNC-Sec61 density and difference map. (PDF 808 kb)

Supplementary Table 1

Statistics of the crystallographic analysis of eEF3. (DOC 31 kb)

Supplementary Table 2

Molecular contact regions between eEF3 and the 80S ribosome. (DOC 38 kb)

Supplementary Notes 1

Supplementary information and comments on Small-angle x-ray scattering (SAXS) experiments. (DOC 31 kb)

Supplementary Notes 2

This file contains additional details of the methods used in this study (X-Ray and SAXS; sample preparation for cryo-EM, EM and processing; docking into EM density) and Supplementary Figures Legends. (DOC 83 kb)

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Andersen, C., Becker, T., Blau, M. et al. Structure of eEF3 and the mechanism of transfer RNA release from the E-site. Nature 443, 663–668 (2006). https://doi.org/10.1038/nature05126

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