Selenocysteine is a unique amino acid that is incorporated specifically into a select group of essential proteins. This process represents a modification of the standard eukaryotic protein synthetic machinery in that it requires the utilization of a novel translation elongation factor (eEFSec), a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region (UTR) of selenoprotein mRNAs, and a novel SECIS binding protein termed SBP2. These factors act in concert to alter the coding potential of specific in-frame stop codons (UGA) by specifying the insertion of selenocysteine at that site. The goal of this project is to understand the interplay between SBP2 and the translation machinery. Since SBP2 is a limiting factor required for Sec incorporation, it is central to the regulation of selenoprotein synthesis, and many selenoproteins are known to play critical roles in cellular defense from oxidation. As a result, SBP2 will be a key target for methods designed to increase the beneficial properties of selenoproteins including the prevention of DNA damage in carcinogenesis and oxidation of pathogenic lipids involved in atherosclerosis. This proposal seeks to understand the function of SBP2 by studying its specific interaction with ribosomes and its interaction with the machinery that would ordinarily dictate the termination of translation at selenocysteine (UGA) codons. Our preliminary studies indicate that SBP2 specifically interacts with ribosomal RNA from yeast to rodents, indicating that the SECIS element may have evolved from highly conserved rRNA structures. This study will combine the utilization of mammalian cells as well as the yeast S. cerevisiae in order to explore this interaction while exploiting the unique benefits of each system. Specifically, we will use mammalian rRNA fragments to probe the binding characteristics of SBP2. This will be integrated with an analysis of SBP2 binding to intact ribosomes using a system unique to yeast by which homogeneous mutant ribosomes can be produced in vivo. Based on the dual function of the UGA stop codon, we will directly assess the role of SBP2 in preventing termination at selenocysteine codons by analyzing selenocysteine incorporation from efficient and inefficient templates. The information we obtain about the function of SBP2 will be used to develop a detailed model for selenocysteine incorporation which should shed light on the basic mechanism of translation termination as well as enable us to identify components of the system that can be manipulated in order to optimize selenoprotein synthesis.
