The long term goal of the research is to understand the molecular mechanisms by which different regulatory pathways interact to control expression of the Neurospora crassa arg-2 gene. The arg-2 gene encodes the mitochondrially localized small subunit of carbamoyl phosphate synthetase. The major goal of the application is to understand the mechanism of arginine-specific, negative translational control mediated by an upstream open reading frame (uORF) in arg-2 mRNA. The arg-2 uORF is essential for translational regulation in response to arginine availability. The primary amino acid sequence of the predicted uORF peptide, which is conserved in other uORFs, is critical for control. Arginine specific translational control through the arg-2 uORF will be assessed in vivo by analyzing levels of RNA and polypeptide synthesis and by examining the distribution of ribosomes on mRNA. Arginine specific translational control will be assessed in vitro using N. crassa cell free translation extracts programmed with synthetic RNAs. There are three specific aims. First, the trans-acting genes that are important for translational control will be identified. Mutants that show effects on arginine-specific regulation will be isolated and characterized. The genes will be placed into complementation groups, mapped, and isolated. The roles of the gene products in arginine-specific translational control will be determined. Second, site-specific mutagenesis will be used to determine the sequence requirements for translational control. The features of both the uORF sequence and the RNA important for translational control will be determined. Third, the mechanism of arginine-specific translational control will be determined. The ability of a synthetic uORF peptide to affect translational processes in trans will be analyzed. The form of arginine that elicits control-free arginine, an arginine metabolite, or charged tRNA-will be determined.
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