This proposal describes experiments to determine the mechanism and prevalence of a new translational initiation process. Translational initiation on most capped eukaryotic mRNAs begins with the recruitment of ribosomal 40S subunits to the 5' end of the mRNA via an interaction of ribosome-associated and cap binding protein complex-associated factors. The 40S subunit then scans the mRNA until an appropriate AUG state codon is encountered, at which a 60S ribosomal subunit joints to assemble a 80S ribosome, with the AUG start codon positioned in the ribosomal P-site. However, certain viral and cellular mRNAs have been identified that contain internal ribosome entry sites (IRES) elements that can direct 40S subunits directly to the mRNA. An IRES element from the intergenic region of critical paralysis virus (IGR-IRES_ directs the formation of initiating 80S ribosomes without initiator tRNAmet/eIF2- GTP complexes of GTP hydrolysis, and translation commences from the A-site of the ribosome, with RNA sequences in the IRES itself interacting at the P-site. Using ribosome assembly assays, toe-printing analysis and chemical and structural probing techniques, we will study the mechanisms by which ribosomal subunits are recruited to the IGR- IRES and by which 80S ribosomes are assembled on the IGR-IRES. The finding that the IGR-IRES is functioning under certain conditions in Saccharomyces cerevisiae opens the possibility to use both biochemical and genetic approaches to study the steps in IRES-mediated translation initiation complexes can be formed on the IGR-IRES when functional initiator tRNAmet/eIF2-GTP complexes are limiting in cultured cells. Finally, using cDNA microarray-based approaches, cellular mRNAs that display IGR-IRES like activities will be isolated and characterized. It is predicted that this unusual translation initiation pathway may operate when cells encounter a variety of stress situations, such as viral infection, heat shock, nutritional deprivation and hypoxia.
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