Nature has allowed the expansion of the genetic code to include two unusual amino acids: selenocysteine (Sec) and pyrrolysine. Interestingly, however, neither of these unique amino acids are utilized by fungi, thus making the yeast Saccharomyces cerevisiae a genetically manipulable blank slate for reconstituting genetic code expansion. This proposal focuses on the hypothesis that reconstitution of Sec incorporation in yeast will dramatically increase our understanding of the mechanism of Sec incorporation as well as allow for the manipulation of this system for the production of site-specifically labeled proteins. The transformation of a UGA stop codon into a Sec codon requires the utilization of a novel translation elongation factor (eEFSec), a selenocysteine insertion sequence (SECIS) element in the 3'untranslated region of selenoprotein mRNAs, and a novel SECIS binding protein termed SBP2. These factors act in concert to alter the coding potential of specific UGA codons by specifying the insertion of the Sec-specific tRNA, Sec-tRNASec. This process is required for the production of 25 human selenoproteins, many of which form an essential line of defense against oxidative stress. In order to rebuild the Sec incorporation system in yeast, we propose to create a series of strains that will allow a stepwise approach to reconstitution. The process will start with Sec- tRNASec, moving up to incorporation of Sec into a luciferase reporter and culminating with a series of genetic screens designed to identify factors that enhance Sec incorporation as well as select for components able to support the site-specific incorporation of unnatural (e.g. fluorescent) amino acids. In addition to the major impact on selenium biology, this project will also provide valuable resources for scientific disciplines that require the analysis of protein structure and function.
This project is designed to reconstitute the utilization of selenium in the form of selenocysteine in the yeast Saccharomyces cerevisiae. The unique molecular machinery that is required for selenocysteine utilization will be manipulated to allow protein engineering. In addition, this system will form the test bed for therapeutics agents designed to regulated the production of selenium-containing proteins in vivo.
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