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 language | English (US) |
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Pages (from-to) | 319-325 |
Number of pages | 7 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 112 |
Issue number | 2 |
DOIs | |
State | Published - Jan 13 2015 |
Externally published | Yes |
Keywords
- HCV IRES
- Human ribosomes
- Single-molecule FRET
- Translation initiation
ASJC Scopus subject areas
- General