Regulation of eukaryotic translation initiation

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DESCRIPTION (provided by applicant): Translation initiation is a multiple-step process involving a large number of eukaryotic initiation factors (elFs) that ultimately orchestrate assembly of the Met-tRNAiMet base-paired with the AUG start codon of mRNA on the 80S ribosome. Our long-term goal is to elucidate the regulation of this process as it plays a critical role in development, differentiation, cell cycle progression, cell growth, and apoptosis and its deregulation results in a loss of cell cycle control and malignant transformation of cells. Of all the initiation factors, elF3 is particularly intriguing because it stimulates multiple steps in the pathway such as binding of Met-tRNAiMet and mRNA to the 40S ribosome. The specific hypothesis is that yeast elF3 promotes 40S-binding of elFs 1, 2, and 5, that were previously implicated in the AUG recognition process, and thus co-regulates the process during which the 40S ribosome scans the 5' UTR of mRNA until it encounters the start AUG codon. We base that hypothesis on the observations that A) yeast elF3 forms a multifactor complex (MFC) with elFs 1, 5 and Met-tRNAiMet-elF2-GTP ternary complex (TC) that can exist free of ribosomes, B) several potential contacts that we found between elF3 and the 40S ribosome seem to be important for the MFC delivery to the ribosome, and C) clustered-alanine mutations in NIP1 subunit of elF3 appear to influence 43S complex formation and stringency of AUG recognition. To address this hypothesis: 1) We will mutate the elF3-40S binding sites and analyze their physiological importance using techniques of yeast genetics and biochemistry, e.g. in vitro binding assays, in vivo affinity chromatography, HCHO cross-linking and fractionation of extracts by sucrose gradients sedimentation; and 2) attempt to identify additional MFC contacts with 40S. 3) We will conduct a site-directed mutagenesis of the selected segments of the NIP1 subunit of elF3 that mediates interactions with elFs 1, 2, and 5 followed by thorough analysis of mutant phenotypes indicating relaxed stringency of AUG selection and defects in TC recruitment to 40S.