Selenium plays a key metabolic role as selenocysteine the 21st amino acid that is encoded by the genetic code. Of the 30-50 selenocysteine containing proteins in mammalian cells, only 10 or so are well characterized. In most cases the amino acid is at the active site and plays a key role in the catalytic reaction and often plays a key role in protection from oxidative insult. Selenocysteine is encoded by UGA, which in most mRNAs is the signal for stop. A complex stem loop in the 3' untranslated region of these special mRNAs (SECIS element) somehow reprograms the ribosome to read UGA as selenocysteine via a tRNA specific for this amino acid. The mechanism of insertion in eukaryotes is not understood though it is clearly different than in prokaryotes. We will use the power of C. elegans genetic system to find the genes involved in the insertion pathway to work out the mammalian homologs. Both up and down the mutants will be sought using a vector with a selectable gene and a GFP screening gene each engineered to have a UGA codon that needs to be read by the selenocysteine insertion mechanism. Mutants will be mapped, their genes identified and products characterized. The structure of the SECIS element and its interactions with any proteins in the pathway will be studied.
Six specific aims are to: (1) establish a C. elegans selection system for up and down mutants of Sec synthesis; (2) map and characterize identified mutant genes; (3) identify interacting components; (4) determine SECIS structure, including using NMR; (5) test RNA binding of putative selB homolog; (6) test models for eukaryotic Se insertion.
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