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Structure of the Escherichia coli ribosomal termination complex with release factor 2

Nature volume 421, pages 90–94 (2003)Cite this article

Abstract

Termination of protein synthesis1 occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs2, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein–transfer-RNA bond hydrolysis3,4. Crystal structures of bacterial and eukaryotic class I RFs have been determined5,6, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the ‘SPF’ loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.

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Figure 1: Stereo views of the structure of the post-termination complex revealing the location of release factor RF2.
Figure 2: Detailed stereo views of RF2–ribosome interactions at the level of the decoding site and the PTC.
Figure 3: The derived in situ RF2 structure and its interaction pattern.

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References

  1. Kisselev, L. L. & Buckingham, R. H. Translational termination comes of age. Trends Biochem. Sci. 25, 561–566 (2000)

    Article  CAS  Google Scholar 

  2. Ito, K., Uno, M. & Nakamura, Y. A tripeptide ‘anticodon’ deciphers stop codons in messenger RNA. Nature 403, 680–684 (2000)

    Article  ADS  CAS  Google Scholar 

  3. Frolova, L. Y. et al. Mutations in the highly conserved GGQ-motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis. RNA 5, 1014–1020 (1999)

    Article  CAS  Google Scholar 

  4. Zavialov, A. V., Mora, L., Buckingham, R. H. & Ehrenberg, M. Release of peptide promoted by the GGQ-motif of class 1 release factors regulates the GTPase activity of RF3. Mol. Cell (in the press)

  5. Song, H. et al. The crystal structure of human eukaryotic release factor eRF1-mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell 100, 311–321 (2000)

    Article  CAS  Google Scholar 

  6. Vestergaard, B. et al. Bacterial polypeptide release factor RF2 is structurally distinct from eukaryotic eRF1. Mol. Cell 8, 1375–1382 (2001)

    Article  CAS  Google Scholar 

  7. Freistroffer, D. V., Pavlov, M. Y., MacDougall, J., Buckingham, R. H. & Ehrenberg, M. Release factor RF3 in E. coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP dependent manner. EMBO J. 16, 4126–4133 (1997)

    Article  CAS  Google Scholar 

  8. Zavialov, A. V., Buckingham, R. H. & Ehrenberg, M. A Posttermination ribosomal complex is the guanine nucleotide exchange factor for peptide release factor RF3. Cell 107, 115–124 (2001)

    Article  CAS  Google Scholar 

  9. van Heel, M. et al. Single-particle electron cryo-microscopy: towards atomic resolution. Q. Rev. Biophys. 33, 307–369 (2000)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  11. Moffat, J. G. & Tate, W. P. A single proteolytic cleavage in release factor 2 stabilizes ribosome binding and abolishes peptidyl-tRNA hydrolysis activity. J. Biol. Chem. 269, 18899–18903 (1994)

    CAS  PubMed  Google Scholar 

  12. Tin, O. F. et al. Proteolytic fragmentation of polypeptide release factor 1 of Thermus thermophilus and crystallization of the stable fragments. Biochimie 82, 765–772 (2000)

    Article  CAS  Google Scholar 

  13. Kastner, B., Trotman, C. N. & Tate, W. P. Localization of the release factor-2 binding site on 70 S ribosomes by immuno-electron microscopy. J. Mol. Biol. 212, 241–245 (1990)

    Article  CAS  Google Scholar 

  14. Wilson, K. S., Ito, K., Noller, H. F. & Nakamura, Y. Functional sites of interaction between release factor RF1 and the ribosome. Nature Struct. Biol. 7, 866–870 (2000)

    Article  CAS  Google Scholar 

  15. Xu, W., Pagel, F. T. & Murgola, E. J. Mutations in the GTPase center of Escherichia coli 23S rRNA indicate release factor 2-interactive sites. J. Bacteriol. 184, 1200–1203 (2002)

    Article  CAS  Google Scholar 

  16. Wimberly, B. T., Guymon, R., McCutcheon, J. P., White, S. W. & Ramakrishnan, V. A detailed view of a ribosomal active site: the structure of the L11–RNA complex. Cell 97, 491–502 (1999)

    Article  CAS  Google Scholar 

  17. Conn, G. L., Draper, D. E., Lattman, E. E. & Gittis, A. G. Crystal structure of a conserved ribosomal protein–RNA complex. Science 284, 1171–1174 (1999)

    Article  ADS  CAS  Google Scholar 

  18. Yusupova, G. Z., Yusupov, M. M., Cate, J. H. & Noller, H. F. The path of messenger RNA through the ribosome. Cell 106, 233–241 (2001)

    Article  CAS  Google Scholar 

  19. Uno, M., Ito, K. & Nakamura, Y. Polypeptide release at sense and noncognate stop codons by localized charge-exchange alterations in translational release factors. Proc. Natl Acad. Sci. USA 99, 1819–1824 (2002)

    Article  ADS  CAS  Google Scholar 

  20. Nissen, P. et al. Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. Science 270, 1464–1472 (1995)

    Article  ADS  CAS  Google Scholar 

  21. Ito, K., Ebihara, K., Uno, M. & Nakamura, Y. Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis. Proc. Natl Acad. Sci. USA 93, 5443–5448 (1996)

    Article  ADS  CAS  Google Scholar 

  22. Bertram, G., Bell, H. A., Ritchie, D. W., Fullerton, G. & Stansfield, I. Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition. RNA 6, 1236–1247 (2000)

    Article  CAS  Google Scholar 

  23. Frolova, L. Y., Seit-Nebi, A. & Kisselev, L. L. Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1. RNA 8, 129–136 (2002)

    Article  CAS  Google Scholar 

  24. Inagaki, Y., Blouin, C., Doolittle, W. F. & Roger, A. J. Convergence and constraint in eukaryotic release factor 1 (eRF1) domain 1: the evolution of stop codon specificity. Nucleic Acids Res. 30, 532–544 (2002)

    Article  CAS  Google Scholar 

  25. Merkulova, T. I., Frolova, L. Y., Lazar, M., Camonis, J. & Kisselev, L. L. C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction. FEBS Lett. 443, 41–47 (1999)

    Article  CAS  Google Scholar 

  26. Jelenc, P. C. & Kurland, C. G. Nucleotide triphosphate regeneration decreases the frequency of translation errors. Proc. Natl Acad. Sci. USA 76, 3174–3178 (1979)

    Article  ADS  CAS  Google Scholar 

  27. Harauz, G. & van Heel, M. Exact filters for general geometry three-dimensional reconstruction. Optik 73, 146–156 (1986)

    Google Scholar 

  28. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  29. Evans, S. V. Setor: Hardware lighted three-dimensional solid model representations of macromolecules. J. Mol. Graphics 11, 134–138 (1993)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Kjeldgaard for making the RF2 coordinates available before deposition; E. Murgola for sharing data before publication; E. Morris, R. Finn and R. Matadeen for discussions on image processing; M. Schatz and R. Schmidt for improvements to the IMAGIC software system; G. Willoughby for computational support; and R. Brimacombe for discussions. This work was supported in part by grants from the BBSRC and the EU. B.V. was funded by the NIH. A.V.Z. and M.E. were supported by the Swedish Foundation for Strategic Research and the Swedish Research Council.

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Author notes

  1. Elena V. Orlova

    Present address: School of Crystallography, Birkbeck College, University of London, London, WC1 7HX, UK

Authors and Affiliations

  1. Department of Biological Sciences, Imperial College of Science, Technology and Medicine, SW7 2AY, London, UK

    Bruno P. Klaholz, Tillmann Pape, Alexander G. Myasnikov, Elena V. Orlova & Marin van Heel

  2. Department of Cell and Molecular Biology, BMC, Uppsala University, Box 596, 75124, Uppsala, Sweden

    Andrey V. Zavialov & Måns Ehrenberg

  3. Institute of Molecular and Structural Biology, University of Aarhus, 8000 C, Aarhus, Denmark

    Bente Vestergaard

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Correspondence to Marin van Heel.

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M.v.H. is a shareholder of Image Science Software GmbH, distributors of the IMAGIC-5 software.

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Klaholz, B., Pape, T., Zavialov, A. et al. Structure of the Escherichia coli ribosomal termination complex with release factor 2. Nature 421, 90–94 (2003). https://doi.org/10.1038/nature01225

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