Studies on start codon selection by eukaryotic ribosomes

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DESCRIPTION (provided by applicant): Translation initiation is the rate-limiting step of protein biosynthesis and an important target for the control of gene expression. Unregulated translation initiation can result in malignant transformation of cells. The yeast Saccharomyces cerevisiae is a genetically tractable eukaryotic microorganism, which has proven to be an excellent model system to study a variety of cellular processes including translation initiation. The aim of this project is to understand at the molecular level, using S. cerevisiae as a model organism, how eukaryotic ribosomal initiation complexes stringently select AUG as a start codon. Extensive genetic and biochemical studies conducted by the Donahue group at the Indiana University implicated eukaryotic initiation factor 1 (elFi), elF5, and all three subunits of elF2 in the stringent AUG selection for yeast mRNAs. elF5 acts as a GTPase activating protein (GAP) for elF2 on correct AUG recognition. Working with Dr. HInnebusch at the NIH, I recently showed that a five-subunit factor elF3 binds to both eIF1 and elF5, and that the C-terminal domain (CTD) of elF5 bridges interaction between elF2 and elF3. The elF1/elF3/elF5/ elF2 multifactor complex occurs free of the ribosome and contains stoichiometric amount of the methionyl initiator tRNA. When bound to the ribosome, the multifactor complex interacts (also via elF5-CTD) with elF4F bound to m7G-capped mRNA. Here I hypothesize that the multiple interactions mediated by the eIF5-CTD stimulate formation of 40S ribosomal preinitiation complexes and also promote scanning and stringent selection of AUG codons. I also propose a model for how the elF2 GTPase is activated on correct AUG recognition via direct elF2-elF5 (GAP) interation. I would like to test these models with mutagenesis studies on elF5 and elF4G, in combination with sophisticated biochemical techniques adapted from studies on mammalian systems.