1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome

  1. Priyanka D Abeyrathne
  2. Cha San Koh
  3. Timothy Grant
  4. Nikolaus Grigorieff
  5. Andrei A Korostelev  Is a corresponding author
  1. Janelia Research Campus, Howard Hughes Medical Institute, United States
  2. University of Massachusetts Medical School, United States
https://doi.org/10.7554/eLife.14874
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Cite this article
  1. Priyanka D Abeyrathne
  2. Cha San Koh
  3. Timothy Grant
  4. Nikolaus Grigorieff
  5. Andrei A Korostelev
(2016)
Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome
eLife 5:e14874.
https://doi.org/10.7554/eLife.14874
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Abstract

Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation.

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Author details

  1. Priyanka D Abeyrathne

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    No competing interests declared.

  2. Cha San Koh

    RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.

  3. Timothy Grant

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    No competing interests declared.

  4. Nikolaus Grigorieff

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    Nikolaus Grigorieff, Reviewing editor, eLife.

  5. Andrei A Korostelev

    RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    andrei.korostelev@umassmed.edu
    Competing interests
    No competing interests declared.

Copyright

© 2016, Abeyrathne et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Priyanka D Abeyrathne
  2. Cha San Koh
  3. Timothy Grant
  4. Nikolaus Grigorieff
  5. Andrei A Korostelev
(2016)
Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome
eLife 5:e14874.
https://doi.org/10.7554/eLife.14874

Share this article

https://doi.org/10.7554/eLife.14874

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    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Structural characterization of ribosome recruitment and translocation by type IV IRES

    Jason Murray, Christos G Savva ... Israel S Fernández
    Research Article

    Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts the otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.