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

Proc Natl Acad Sci U S A. 2015 Jan 13;112(2):319-25. doi: 10.1073/pnas.1421328111. Epub 2014 Dec 16.

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.

Keywords: HCV IRES; human ribosomes; single-molecule FRET; translation initiation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites / genetics
  • CRISPR-Cas Systems
  • Fluorescence Resonance Energy Transfer
  • Gene Knockout Techniques
  • HeLa Cells
  • Hepacivirus / genetics*
  • Hepacivirus / metabolism*
  • Hepacivirus / pathogenicity
  • Host-Pathogen Interactions
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • O(6)-Methylguanine-DNA Methyltransferase / genetics
  • O(6)-Methylguanine-DNA Methyltransferase / metabolism
  • Peptide Chain Initiation, Translational
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Ribosomal Proteins / antagonists & inhibitors
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / metabolism
  • Ribosome Subunits, Small, Eukaryotic / metabolism*

Substances

  • RNA, Messenger
  • RNA, Viral
  • RPS25 protein, human
  • Recombinant Fusion Proteins
  • Ribosomal Proteins
  • O(6)-Methylguanine-DNA Methyltransferase