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

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.