title: Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.
abstract: Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124;
authors.forename: Gabriele
authors.initials: G
authors.lastname: Fuchs
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126; and.
authors.forename: Alexey N
authors.initials: AN
authors.lastname: Petrov
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124;
authors.forename: Caleb D
authors.initials: CD
authors.lastname: Marceau
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124;
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Popov
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126; and Department of Applied Physics, Stanford University, Stanford, CA 94305-4090.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126; and.
authors.forename: Seán E
authors.initials: SE
authors.lastname: O'Leary
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126; and.
authors.forename: Richard
authors.initials: R
authors.lastname: Wang
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124;
authors.forename: Jan E
authors.initials: JE
authors.lastname: Carette
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124;
authors.forename: Peter
authors.initials: P
authors.lastname: Sarnow
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126; and puglisi@stanford.edu.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D064113:CRISPR-Cas Systems; D031541:Fluorescence Resonance Energy Transfer; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism* / Q000472:pathogenicity; D054884:Host-Pathogen Interactions; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D010442:Peptide Chain Initiation, Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral; C489168:RPS25 protein, human; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase
keywords: HCV IRES; human ribosomes; single-molecule FRET; translation initiation