Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structure of the mammalian 80S initiation complex with initiation factor 5B on HCV-IRES RNA

Nature Structural & Molecular Biology volume 21, pages 721–727 (2014)Cite this article

Subjects

Abstract

The universally conserved eukaryotic initiation factor (eIF) 5B, a translational GTPase, is essential for canonical translation initiation. It is also required for initiation facilitated by the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) RNA. eIF5B promotes joining of 60S ribosomal subunits to 40S ribosomal subunits bound by initiator tRNA (Met-tRNAiMet). However, the exact molecular mechanism by which eIF5B acts has not been established. Here we present cryo-EM reconstructions of the mammalian 80S–HCV-IRES–Met-tRNAiMet–eIF5B–GMPPNP complex. We obtained two substates distinguished by the rotational state of the ribosomal subunits and the configuration of initiator tRNA in the peptidyl (P) site. Accordingly, a combination of conformational changes in the 80S ribosome and in initiator tRNA facilitates binding of the Met-tRNAiMet to the 60S P site and redefines the role of eIF5B as a tRNA-reorientation factor.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cryo-EM reconstructions of the 80S–HCV-IRES–Met-tRNAiMet–eIF5B–GMPPNP complex.
Figure 2: 40S-subunit rolling rearranges initiator tRNA in the Pre- and Post-like states.
Figure 3: Interaction of eIF5B with the ribosome in the Pre- and Post-like states.
Figure 4: Movement of HCV-IRES domain II.
Figure 5: Subunit joining requires adjustment of initiator tRNA in the peptidyl transferase cleft of the 60S between the central protuberance and the H69 rim.
Figure 6: The trajectory of initiator tRNA suggests a model in which eIF5B acts as a tRNA-reorientation factor in ribosomal subunit joining.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Referenced accessions

Electron Microscopy Data Bank

Protein Data Bank

References

  1. Aitken, C.E. & Lorsch, J.R. A mechanistic overview of translation initiation in eukaryotes. Nat. Struct. Mol. Biol. 19, 568–576 (2012).

    Article  CAS  Google Scholar 

  2. Pestova, T.V. et al. The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 403, 332–335 (2000).

    Article  CAS  Google Scholar 

  3. Choi, S.K., Lee, J.H., Zoll, W.L., Merrick, W.C. & Dever, T.E. Promotion of met-tRNAiMet binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast. Science 280, 1757–1760 (1998).

    Article  CAS  Google Scholar 

  4. Wilson, S.A. et al. Cloning and characterization of hIF2, a human homologue of bacterial translation initiation factor 2, and its interaction with HIV-1 matrix. Biochem. J. 342, 97–103 (1999).

    Article  CAS  Google Scholar 

  5. Carrera, P. et al. VASA mediates translation through interaction with a Drosophila yIF2 homolog. Mol. Cell 5, 181–187 (2000).

    Article  CAS  Google Scholar 

  6. Strunk, B.S., Novak, M.N., Young, C.L. & Karbstein, K. A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits. Cell 150, 111–121 (2012).

    Article  CAS  Google Scholar 

  7. Pestova, T.V. et al. Molecular mechanisms of translation initiation in eukaryotes. Proc. Natl. Acad. Sci. USA 98, 7029–7036 (2001).

    Article  CAS  Google Scholar 

  8. Roll-Mecak, A., Cao, C., Dever, T.E. & Burley, S.K. X-ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding. Cell 103, 781–792 (2000).

    Article  CAS  Google Scholar 

  9. Unbehaun, A. et al. Position of eukaryotic initiation factor eIF5B on the 80S ribosome mapped by directed hydroxyl radical probing. EMBO J. 26, 3109–3123 (2007).

    Article  CAS  Google Scholar 

  10. Pisarev, A.V. et al. Specific functional interactions of nucleotides at key –3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex. Genes Dev. 20, 624–636 (2006).

    Article  CAS  Google Scholar 

  11. Lee, J.H., Choi, S.K., Roll-Mecak, A., Burley, S.K. & Dever, T.E. Universal conservation in translation initiation revealed by human and archaeal homologs of bacterial translation initiation factor IF2. Proc. Natl. Acad. Sci. USA 96, 4342–4347 (1999).

    Article  CAS  Google Scholar 

  12. Lee, J.H. et al. Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation. Proc. Natl. Acad. Sci. USA 99, 16689–16694 (2002).

    Article  CAS  Google Scholar 

  13. Shin, B.S. et al. Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity. Cell 111, 1015–1025 (2002).

    Article  CAS  Google Scholar 

  14. Pestova, T.V., de Breyne, S., Pisarev, A.V., Abaeva, I.S. & Hellen, C.U. eIF2-dependent and eIF2-independent modes of initiation on the CSFV IRES: a common role of domain II. EMBO J. 27, 1060–1072 (2008).

    Article  CAS  Google Scholar 

  15. Terenin, I.M., Dmitriev, S.E., Andreev, D.E. & Shatsky, I.N. Eukaryotic translation initiation machinery can operate in a bacterial-like mode without eIF2. Nat. Struct. Mol. Biol. 15, 836–841 (2008).

    Article  CAS  Google Scholar 

  16. Locker, N., Easton, L.E. & Lukavsky, P.J. HCV and CSFV IRES domain II mediate eIF2 release during 80S ribosome assembly. EMBO J. 26, 795–805 (2007).

    Article  CAS  Google Scholar 

  17. Lukavsky, P.J. Structure and function of HCV IRES domains. Virus Res. 139, 166–171 (2009).

    Article  CAS  Google Scholar 

  18. Pestova, T.V., Borukhov, S.I. & Hellen, C.U. Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons. Nature 394, 854–859 (1998).

    Article  CAS  Google Scholar 

  19. Allen, G.S., Zavialov, A., Gursky, R., Ehrenberg, M. & Frank, J. The cryo-EM structure of a translation initiation complex from Escherichia coli. Cell 121, 703–712 (2005).

    Article  CAS  Google Scholar 

  20. Myasnikov, A.G. et al. Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome. Nat. Struct. Mol. Biol. 12, 1145–1149 (2005).

    Article  CAS  Google Scholar 

  21. Shin, B.S. et al. rRNA suppressor of a eukaryotic translation initiation factor 5B/initiation factor 2 mutant reveals a binding site for translational GTPases on the small ribosomal subunit. Mol. Cell. Biol. 29, 808–821 (2009).

    Article  CAS  Google Scholar 

  22. Fernández, I.S. et al. Molecular architecture of a eukaryotic translational initiation complex. Science 342, 1240585 (2013).

    Article  Google Scholar 

  23. Loerke, J., Giesebrecht, J. & Spahn, C.M. Multiparticle cryo-EM of ribosomes. Methods Enzymol. 483, 161–177 (2010).

    Article  CAS  Google Scholar 

  24. Scheres, S.H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).

    Article  CAS  Google Scholar 

  25. Budkevich, T. V. et al. Regulation of the mammalian elongation cycle by 40S subunit rolling: a eukaryotic specific ribosome rearrangement. Cell 158, 121–131 (2014).

    Article  CAS  Google Scholar 

  26. Hashem, Y. et al. Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit. Nature 503, 539–543 (2013).

    Article  CAS  Google Scholar 

  27. Unbehaun, A., Borukhov, S.I., Hellen, C.U. & Pestova, T.V. Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP. Genes Dev. 18, 3078–3093 (2004).

    Article  CAS  Google Scholar 

  28. Budkevich, T. et al. Structure and dynamics of the mammalian ribosomal pretranslocation complex. Mol. Cell 44, 214–224 (2011).

    Article  CAS  Google Scholar 

  29. Guillon, L., Schmitt, E., Blanquet, S. & Mechulam, Y. Initiator tRNA binding by e/aIF5B, the eukaryotic/archaeal homologue of bacterial initiation factor IF2. Biochemistry 44, 15594–15601 (2005).

    Article  CAS  Google Scholar 

  30. Ratje, A.H. et al. Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites. Nature 468, 713–716 (2010).

    Article  CAS  Google Scholar 

  31. 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 

  32. Kuhle, B. & Ficner, R. eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining. EMBO J. 33, 1177–1191 (2014).

    Article  CAS  Google Scholar 

  33. Spahn, C.M.T. et al. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40S ribosomal subunit. Science 291, 1959–1962 (2001).

    Article  CAS  Google Scholar 

  34. Filbin, M.E., Vollmar, B.S., Shi, D., Gonen, T. & Kieft, J.S. HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation. Nat. Struct. Mol. Biol. 20, 150–158 (2013).

    Article  CAS  Google Scholar 

  35. Sprang, S.R. G protein mechanisms: insights from structural analysis. Annu. Rev. Biochem. 66, 639–678 (1997).

    Article  CAS  Google Scholar 

  36. Voorhees, R.M. & Ramakrishnan, V. Structural basis of the translational elongation cycle. Annu. Rev. Biochem. 82, 203–236 (2013).

    Article  CAS  Google Scholar 

  37. 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 

  38. Villa, E. et al. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc. Natl. Acad. Sci. USA 106, 1063–1068 (2009).

    Article  CAS  Google Scholar 

  39. Hiraishi, H. et al. Interaction between 25S rRNA A loop and eukaryotic translation initiation factor 5B promotes subunit joining and ensures stringent AUG selection. Mol. Cell. Biol. 33, 3540–3548 (2013).

    Article  CAS  Google Scholar 

  40. Acker, M.G. et al. Kinetic analysis of late steps of eukaryotic translation initiation. J. Mol. Biol. 385, 491–506 (2009).

    Article  CAS  Google Scholar 

  41. Marshall, R.A., Aitken, C.E. & Puglisi, J.D. GTP hydrolysis by IF2 guides progression of the ribosome into elongation. Mol. Cell 35, 37–47 (2009).

    Article  CAS  Google Scholar 

  42. Shin, B.S. et al. Structural integrity of α-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability. RNA 17, 687–696 (2011).

    Article  CAS  Google Scholar 

  43. Lomakin, I.B. & Steitz, T.A. The initiation of mammalian protein synthesis and mRNA scanning mechanism. Nature 500, 307–311 (2013).

    Article  CAS  Google Scholar 

  44. Budkevich, T.V., El′skaya, A.V. & Nierhaus, K.H. Features of 80S mammalian ribosome and its subunits. Nucleic Acids Res. 36, 4736–4744 (2008).

    Article  CAS  Google Scholar 

  45. Namy, O., Moran, S.J., Stuart, D.I., Gilbert, R.J. & Brierley, I. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting. Nature 441, 244–247 (2006).

    Article  CAS  Google Scholar 

  46. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    Article  CAS  Google Scholar 

  47. 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 

  48. Chen, J.Z. & Grigorieff, N. SIGNATURE: A single-particle selection system for molecular electron microscopy. J. Struct. Biol. 157, 168–173 (2007).

    Article  CAS  Google Scholar 

  49. 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 

  50. 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 

  51. Hohn, M. et al. SPARX, a new environment for cryo-EM image processing. J. Struct. Biol. 157, 47–55 (2007).

    Article  CAS  Google Scholar 

  52. Yang, Z. & Penczek, P.A. Cryo-EM image alignment based on nonuniform fast Fourier transform. Ultramicroscopy 108, 959–969 (2008).

    Article  CAS  Google Scholar 

  53. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  54. Keating, K.S. & Pyle, A.M. RCrane: semi-automated RNA model building. Acta Crystallogr. D Biol. Crystallogr. 68, 985–995 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  56. Brunger, A.T. Version 1.2 of the Crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

    Article  CAS  Google Scholar 

  57. Laurberg, M. et al. Structural basis for translation termination on the 70S ribosome. Nature 454, 852–857 (2008).

    Article  CAS  Google Scholar 

  58. Buchan, D.W., Minneci, F., Nugent, T.C., Bryson, K. & Jones, D.T. Scalable web services for the PSIPRED Protein Analysis Workbench. Nucleic Acids Res. 41, W349–W357 (2013).

    Article  Google Scholar 

  59. Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T.V. Pestova (SUNY Downstate Medical Center) for expression vectors of eIF5B587–1220 (ΔeIF5B) and of Escherichia coli methionyl-tRNA synthetase, P. Lukavsky (ETH Zürich), for the HCV-IRES construct, T. Budkevich for help with tRNA purification, J. Ismer for help with modeling of the N-terminal part of eIF5B, C. Lally for proofreading of the manuscript and K. Yamamoto for helpful discussion. This work was supported by a grant from the German Research Foundation (DFG; SFB 740, Forschergruppe 1805) to C.M.T.S. A.U. acknowledges a Rahel Hirsch fellowship from the Charité Universitätsmedizin Berlin.

Author information

Author notes

  1. Hiroshi Yamamoto and Anett Unbehaun: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Medical Physics and Biophysics, Charité–Universitätsmedizin, Berlin, Germany

    Hiroshi Yamamoto, Anett Unbehaun, Justus Loerke, Elmar Behrmann, Marianne Collier, Jörg Bürger, Thorsten Mielke & Christian M T Spahn

  2. UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Berlin, Germany

    Jörg Bürger & Thorsten Mielke

Authors
  1. Hiroshi Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anett Unbehaun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Justus Loerke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elmar Behrmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marianne Collier
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jörg Bürger
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Thorsten Mielke
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christian M T Spahn
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.U. and H.Y. established the in vitro system for the reconstitution of initiation complexes. H.Y. prepared the 80S–HCV-IRES–Met-tRNAiMet–eIF5B–GMPPNP complex. H.Y., J.B. and T.M. collected cryo-EM data. H.Y., J.L., E.B. and C.M.T.S. processed images. H.Y. and E.B. did the modeling. M.C. built the model of HCV-IRES RNA. H.Y., A.U. and C.M.T.S. discussed results and wrote the paper.

Corresponding author

Correspondence to Christian M T Spahn.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Multiparticle refinement for 80S–HCV-IRES–Met-tRNAiMet–eIF5B–GMPPNP complex.

Multi-particle refinement was used to overcome heterogeneity of the complex. The 541,570 particle images were analyzed with six-fold decimated data with a pixel size of 4.74 Å (pixel size (PS) 4.74), yielding one major population (267,528; 49 % of the complete data set). This population was further separated into three sub-populations using four-fold decimated data with a pixel size of 3.16 Å (PS 3.16). The population containing HCV IRES, tRNA and eIF5B (182,925; 33 %) was isolated. The isolated polupation was further split into structures correspoiding to a rolled (94,026; 16 %) and an unrolled state (88,899; 19 %). The focussed reassignment for ligands (eIF5B, P- and E-site tRNA) separated the dataset into 6 populations. Two populations (52,930; 9 % and 35,261; 6 %) identified by the absence of E-site tRNA were subsequently refined individually. The isolated populations representing substates I and II (34,388; 6 % and 24,777; 4 %, respectively) from three-fold decimated data with a pixel size of 2.37 Å (PS 2.37) were considered as final maps. No apparent density is present for eIF3, which is known to be released during eIF5B-promoted subunit joining on 80S initiation complexes on cellular and HCV-IRES mRNAs16-18.

Supplementary Figure 2 Resolution curves of the Pre- and Post-like complexes.

Resolution curves for the cryo-EM maps of the PRE-like and POST-like complex of the 80S•HCV-IRES•Met-tRNAi Met•eIF5B•GMPPNP initiation complex. The final resolution for the PRE- and POST-like complex is (a) 8.9 and 9.5 Å, respectively and was estimated using a cutoff of 0.5 in the Fourier shell correlation (FSC) standard curves, (b) 8.2 Å and 8.6 Å using with gold-standard approach using the 0.143 FSC criteria.

Supplementary Figure 3 Conformational change in tRNA upon binding by eIF5B on the ribosome.

(a) Superpostion of PI and P/P tRNAs in a common 40S alignment of the PRE-like state. Experimental map (semi-transparent green and red for tRNA and eIF5B map, respectively) with docked models of PI-tRNA, green and eIF5B domain IV, red), P/P-tRNA from POST state (PDB code 4CXB)19 (gray mesh map and model). (b) Comparison of the PRE-like state (colour) and 40S subunit from the yeast initiation 80S complex (EMD-2422)20 (gray) in a common 60S alignment. (c) Comparison of the Met-tRNAiMet•eIF5B•GMPPNP complex (colored as above) within the mammalian PRE-like state and the yeast initiation 80S complex (gray) (PDB code 4BYX) in a common large subunit alignment.

Supplementary Figure 4 The interaction of the G domain, switch I and domain IV of eIF5B with the ribosome.

The observed interactions are presented as density with fitted models. (a,c,e,g) PRE-like state, (b,d,f,h) POST like state: reconstructed cryo EM density is presented as gray mesh and docked models in color: GMPPNP (cyan), eIF5B (red), 28S rRNA (blue), 60S proteins (orange), 18S rRNA (yellow), 40S proteins (gray).

Supplementary Figure 5 Subunit association of vacant 40S and 60S subunits in the presence or absence of eIF5B.

(a and b) 40S and 60S subunits were incubated without eIF5B or in the presence of eIF5B587-1220 (eIFΔ5B), GTP or GMPPNP, as indicated. Assembly of 80S complexes was analyzed by sucrose gradient centrifugation and continuous OD measurement at 260 nm. The position of 80S complexes, 40S and free 60S subunits are indicated and were identified by comparison with control runs (data not shown). In the presence of eIFΔ5B and GTP or GMPPNP, the position of the peak of free 40S subunit is slightly shifted to the heavier fractions. Subunit joining and gradient centrifugation were performed at physiological and elevated concentration of MgCl2 (a: 2.5 mM MgCl2;b: 5 mM MgCl2).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Note (PDF 72835 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yamamoto, H., Unbehaun, A., Loerke, J. et al. Structure of the mammalian 80S initiation complex with initiation factor 5B on HCV-IRES RNA. Nat Struct Mol Biol 21, 721–727 (2014). https://doi.org/10.1038/nsmb.2859

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2859

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing