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

<div class='caption-title'>(a) Structures I through V. In all panels, the large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. Nucleotides C1274, U1191 of the 40S head and G904 of the platform (C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. Unresolved regions of the IRES in densities for Structures III and V are shown in gray. (b) Schematic representation of the structures shown in panel a, denoting the conformations of the small subunit relative to the large subunit. A, P and E sites are shown as rectangles. All measurements are relative to the non-rotated 80S•2tRNA•mRNA structure (Svidritskiy et al., 2014). The colors are as in panel a.</div>
<div class='caption-title'>(a) Structures of bacterial 70S•2tRNA•mRNA translocation complexes, ordered according to the position of the translocating A->P tRNA (orange). The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body), elongation factor G (EF-G) is shown in green. Nucleotides C1054, G966 and G693 of 16S rRNA are shown in black to denote the A, P and E sites, respectively. The extents of the 30S subunit rotation and head swivel relative to their positions in the post-translocation structure (Gao et al., 2009) are shown with arrows. References and PDB codes of the structures are shown. (b) Structures of the 80S•IRES complexes in the absence (Koh et al., 2014) and presence of eEF2 (this work). The large ribosomal subunit is shown in cyan; the small subunit in light yellow (head) and wheat-yellow (body); the TSV IRES in red, eEF2 in green. Nucleotides C1274, U1191 of the 40S head and G904 of the platform (corresponding to C1054, G966 and G693 in E. coli 16S rRNA) are shown in black to denote the A, P and E sites, respectively. Unresolved regions of the IRES in densities for Structures III and V are shown in gray. The extents of the 40S subunit rotation and head swivel relative to their positions in the post-translocation structure (Svidritskiy et al., 2014) are shown with arrows.</div>
<div class='caption-title'>All particles were initially aligned to a single model. 3D classification using a 3D mask around the 40S head, TSV IRES and eEF2, of the 4x binned stack was used to identify particles containing both the IRES and eEF2. Subsequent 3D classification using a 2D mask comprising PKI and domain IV of eEF2 yielded 5 'purified' classes representing Structures I through V. Sub-classification of each class did not yield additional classes, but helped improve density in the PKI region of class III (estimated resolution and percentage of particles in the sub-classified reconstruction are shown in parentheses).</div>
Abeyrathne PD, Koh CS, Grant T, Grigorieff N, Korostelev AA
OpenAccess logo Elife 5 (2016)
Related entries: 5juo, 5jup, 5jut, 5juu

Cited: 68 times
EuropePMC logo PMID: 27159452

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.

Articles - 5jus mentioned but not cited (2)

  1. A Ribosome Interaction Surface Sensitive to mRNA GCN Periodicity. Scopino K, Williams E, Elsayed A, Barr WA, Krizanc D, Thayer KM, Weir MP. Biomolecules 10 E849 (2020)
  2. RIBFIND2: Identifying rigid bodies in protein and nucleic acid structures. Malhotra S, Mulvaney T, Cragnolini T, Sidhu H, Joseph AP, Beton JG, Topf M. Nucleic Acids Res 51 9567-9575 (2023)


Reviews citing this publication (19)

  1. Challenges and opportunities in cryo-EM single-particle analysis. Lyumkis D. J Biol Chem 294 5181-5197 (2019)
  2. Viral RNA structure-based strategies to manipulate translation. Jaafar ZA, Kieft JS. Nat Rev Microbiol 17 110-123 (2019)
  3. Translation Elongation and Recoding in Eukaryotes. Dever TE, Dinman JD, Green R. Cold Spring Harb Perspect Biol 10 a032649 (2018)
  4. Insights into Structural and Mechanistic Features of Viral IRES Elements. Martinez-Salas E, Francisco-Velilla R, Fernandez-Chamorro J, Embarek AM. Front Microbiol 8 2629 (2017)
  5. Ribosomal Chamber Music: Toward an Understanding of IRES Mechanisms. Yamamoto H, Unbehaun A, Spahn CMT. Trends Biochem Sci 42 655-668 (2017)
  6. Viral internal ribosomal entry sites: four classes for one goal. Mailliot J, Martin F. Wiley Interdiscip Rev RNA 9 (2018)
  7. RNA modifications and cancer. Haruehanroengra P, Zheng YY, Zhou Y, Huang Y, Sheng J. RNA Biol 17 1560-1575 (2020)
  8. Roles of Elongator Dependent tRNA Modification Pathways in Neurodegeneration and Cancer. Hawer H, Hammermeister A, Ravichandran KE, Glatt S, Schaffrath R, Klassen R. Genes (Basel) 10 E19 (2018)
  9. Identifying and Visualizing Macromolecular Flexibility in Structural Biology. Palamini M, Canciani A, Forneris F. Front Mol Biosci 3 47 (2016)
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  12. Computational methods for analyzing conformational variability of macromolecular complexes from cryo-electron microscopy images. Jonić S. Curr Opin Struct Biol 43 114-121 (2017)
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  15. The Structural Dynamics of Translation. Korostelev AA. Annu Rev Biochem 91 245-267 (2022)
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  17. How villains are made: The translation of dipeptide repeat proteins in C9ORF72-ALS/FTD. Van't Spijker HM, Almeida S. Gene 858 147167 (2023)
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Articles citing this publication (47)



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