the discovery that selenocystein is encoded by UGA codons in both prokaryotic and eukaryotic proteins, and that UGA codons specifying both selenocysteine and termination are present in the same cells, let to the fundamental question: How is UGA encoding selenocysteine distinguished from UGA specifying termination? Because the UGA codons in mRNAs encoding seleoproteins are translated with fidelity, and those specifying stop are recognized as such, there must exist features of the mRNAs themselves that allow the translational apparatus to distinguish between these two functions. Putative stem-loops in the 3' untranslated regions of the eukaryotic seleoproteins, iodothyroinine deiodinase, glutathione peroxidase, and selenoprotein P are required for UGA codon recognition. Cellular factors, which may be protein, RNA, or a combination of the two, must somehow recognize these structures and facilitate binding of the selenocysteyl-tRNAuca to the ribosome. The purpose of the proposed studies is to identify and characterize the cellular components responsible for this recognition process, and the specific features which they recognize, with the long-term goal of understanding the mechanism of this critical process. The site of regulation of selenoprotein mRNA levels by selenium will also be investigated. Initial studies have focused on characterizing the mRNA sequences and secondary structures required for selenocysteine insertion. The cellular factors which interact with these sequences or structures will be studies using a variety of methods, including RNA-protein gel mobility shift assays, UV crosslinking, avidin-biotin RNA affinity chromatography, and Northwestern blotting. The selenocysteine insertion RNA sequences will be used as probes to screen expression libraries (Northwestern screening) for proteins which specially bind to them, following methods developed for cloning of sequence-specific DNA-binding proteins. Selenium deficiency is associated with an often fatal cardiomyopathy in China, Keshan disease. Selenocysteine is required for optimal conversion of thyroxine to 3,5,3'-triiodothyronine, the first step in thyroid hormone action. High levels of selenium have been found to produce developmental disorders in birds. The effects of selenium deficiency and toxicity underscore the importance of this essential trace element.
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