title: Structural characterization of ribosome recruitment and translocation by type IV IRES.
abstract: 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 tvhe 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.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.|Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors.forename: Jason
authors.initials: J
authors.lastname: Murray
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Christos G
authors.identifier: 0000-0003-3354-6483
authors.initials: CG
authors.lastname: Savva
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors.forename: Byung-Sik
authors.initials: BS
authors.lastname: Shin
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Dever
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: V
authors.initials: V
authors.lastname: Ramakrishnan
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Israel S
authors.identifier: 0000-0001-7218-1603
authors.initials: IS
authors.lastname: Fernández
mesh_terms: D020285:Cryoelectron Microscopy; D056629:Dicistroviridae / Q000737:chemistry; D000067556:Internal Ribosome Entry Sites; D007716:Kluyveromyces / Q000737:chemistry; D046911:Macromolecular Substances / Q000378:metabolism / Q000648:ultrastructure; D010442:Peptide Chain Initiation, Translational; D020652:Peptide Elongation Factor 2 / Q000378:metabolism / Q000648:ultrastructure; D012333:RNA, Messenger / Q000378:metabolism* / Q000648:ultrastructure; D012367:RNA, Viral / Q000378:metabolism* / Q000648:ultrastructure; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000067556:Internal Ribosome Entry Sites; D046911:Macromolecular Substances; D020652:Peptide Elongation Factor 2; D012333:RNA, Messenger; D012367:RNA, Viral
keywords: IRES; S. cerevisiae; biochemistry; biophysics; cricket; paralysis; ribosome; structural biology; translocation; virus infection