Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site

• Published in: Proceedings of the National Academy of Sciences - PNAS 2015-01, Vol.112 (2), p.319-325
• Main Authors: Fuchs, Gabriele, Petrov, Alexey N., Marceau, Caleb D., Popov, Lauren M., Chen, Jin, O’Leary, Seán E., Wang, Richard, Carette, Jan E., Sarnow, Peter, Puglisi, Joseph D.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Binding sites; Binding Sites - genetics; Biological Sciences; Cells; CRISPR-Cas Systems; Fluorescence Resonance Energy Transfer; Gene Knockout Techniques; HeLa Cells; Hepacivirus - genetics; Hepacivirus - metabolism; Hepacivirus - pathogenicity; Hepatitis; Hepatitis C; Hepatitis C virus; Host-Pathogen Interactions; Humans; INAUGURAL ARTICLE; Kinetics; Molecular Sequence Data; Molecules; O-Methylguanine-DNA Methyltransferase - genetics; O-Methylguanine-DNA Methyltransferase - metabolism; Peptide Chain Initiation, Translational; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Ribosomal Proteins - antagonists & inhibitors; Ribosomal Proteins - genetics; Ribosomal Proteins - metabolism; Ribosome Subunits, Small, Eukaryotic - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Viral - genetics; RNA, Viral - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1421328111

Significance Protein biosynthesis is most tightly controlled during translation initiation that involves numerous initiation factors and regulatory proteins. This complexity confounds conventional biochemical methods. Single-molecule approaches are ideally suited to address such questions. However, their application is hindered by the lack of fluorescently labeled components of the eukaryotic translation machinery. Here, we demonstrate an approach to label human 40S ribosomal subunits. As an extension of this approach, we used single-molecule fluorescence to demonstrate that 40S ribosomal subunits are recruited to the hepatitis C virus mRNA in a single-step process, and that components of a translational extract regulate the conformation of this complex. 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 Fáörster 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.