title: Multiple Parallel Pathways of Translation Initiation on the CrPV IRES.
abstract: 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.
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
authors.forename: Alexey
authors.initials: A
authors.lastname: Petrov
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
authors.forename: Rosslyn
authors.initials: R
authors.lastname: Grosely
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA; Department of Applied Physics, Stanford University, Stanford, CA 94305-4090, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
authors.forename: Seán E
authors.initials: SE
authors.lastname: O'Leary
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA. Electronic address: puglisi@stanford.edu.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D056629:Dicistroviridae / Q000235:genetics* / Q000378:metabolism; D000067556:Internal Ribosome Entry Sites; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism
chemical_list: D000067556:Internal Ribosome Entry Sites; D012367:RNA, Viral; D012269:Ribosomal Proteins; D012343:RNA, Transfer