Multiple Parallel Pathways of Translation Initiation on the CrPV IRES

The complexity of eukaryotic translation allows fine-tuned regulation of protein synthesis. Viruses use internal ribosome entry sites (IRESs) to minimize or, like the CrPV IRES, eliminate the need for initiation factors. Here, by exploiting the CrPV IRES, we observed the entire process of initiation and transition to elongation in real time. We directly tracked the CrPV IRES, 40S and 60S ribosomal subunits, and tRNA using single-molecule fluorescence spectroscopy and identified multiple parallel initiation pathways within the system. Our results distinguished two pathways of 80S:CrPV IRES complex assembly that produce elongation-competent complexes. Following 80S assembly, the requisite eEF2-mediated translocation results in an unstable intermediate that is captured by binding of the elongator tRNA. Whereas initiation can occur in the 0 and +1 frames, the arrival of the first tRNA defines the reading frame and strongly favors 0 frame initiation. Overall, even in the simplest system, an intricate reaction network regulates translation initiation. [Display omitted] •The CrPV IRES can recruit the 40S and 60S subunits sequentially or simultaneously•Both ribosomal recruitment pathways form elongation-competent 80S:IRES complexes•eEF2-mediated translocation results in an unstable intermediate captured by tRNA•80S:CrPV IRES reading frame is selected by 0 and +1 frame tRNA arrival kinetics Petrov et al. observe translation of the CrPV IRES at single-molecule resolution, tracking initiation, elongation, and the transition between these processes. The IRES harnesses multiple pathways to hijack the host translation system, and dynamics define the outcome.