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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

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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.

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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.

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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.

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  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

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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.

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Correspondence to Christian M T Spahn.

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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).

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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

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