The pathway of HCV mRNA recruitment to the human ribosome

Eukaryotic protein synthesis begins with mRNA positioning in the ribosomal decoding channel in a process typically controlled by translation initiation factors. Some viruses utilize an internal ribosome entry site (IRES) in their mRNA to harness ribosomes independently of initiation factors. We show here that a ribosome conformational change induced upon Hepatitis C viral IRES binding is necessary but not sufficient for correct mRNA positioning. Using directed hydroxyl radical probing to monitor the assembly of IRES-containing translation initiation complexes, a critical step has been defined in which mRNA is stabilized upon initiator tRNA binding. Surprisingly, however, this stabilization occurs independent of the AUG codon, underscoring the importance of initiation factor-mediated interactions that influence decoding channel configuration. These results reveal how an IRES RNA supplants some, but not all, of the functions normally carried out by protein factors during protein synthesis initiation.