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

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

Author information

1
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124;
2
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126; and.
3
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126; and Department of Applied Physics, Stanford University, Stanford, CA 94305-4090.
4
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126; and puglisi@stanford.edu.

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

PMID:
25516984
PMCID:
PMC4299178
DOI:
10.1073/pnas.1421328111
[Indexed for MEDLINE]
Free PMC Article

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