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

Gabriele Fuchs, Alexey N. Petrov, Caleb D. Marceau, Lauren M. Popov, Jin Chen, Seán E. OLeary, Richard Wang, Jan E. Carette, Peter Sarnow, Joseph D. Puglisi

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)319-325
Number of pages7
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number2
DOIs
StatePublished - Jan 13 2015
Externally publishedYes

Keywords

  • HCV IRES
  • Human ribosomes
  • Single-molecule FRET
  • Translation initiation

ASJC Scopus subject areas

  • General

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