Excesses and deficiencies of selenium in the diet are detrimental to health and evidence suggests that this element has a role in cancer, heart disease and in the aging process. Since selenium exerts its effects, at least in part, through its presence in protein, our goal is to understand how this fascinating element is incorporated into protein. Thus, we are studying the tRNAs involved in donating selenium in the form of selenocysteine into protein. In the past year, we have shown that the primary sequence of the rat liver selenocysteine tRNAs are colinear with the corresponding selenocysteine tRNA gene sequence and that the structures of these isoacceptors differ by a single 2'-0-ribose methylation in the wobble position of the anticodon. The methylated nucleoside is a newly discovered moiety, not observed in any other tRNA. There are three additional modified nucleosides in these tRNAs. Xenopus selenocysteine tRNA also contains only four modified nucleosides and, interestingly, these are the same four modified moieties found in the rat liver species. Biosynthesis of the selenocysteine tRNAs in Xenopus oocytes from either the gene or from synthetic RNA (generated from the gene cloned into an expression vector) shows that the four modified nucleosides are formed and that a shift in the selenocysteine tRNA population toward the methylated derivative occurs in response to selenium. A ribosomal binding assay was devised which demonstrated that selenocysteyl-tRNA does not interact with the normal elongation factor, EFalpha-1, providing evidence that this unique tRNA has its own elongation factor. Interestingly, the seryl-tRNA form of selenocysteine tRNA did interact, albeit weakly, with EFalpha-1 which substantiated our earlier findings that this tRNA can suppress UGA codons in protein synthesis. Identity of the selenocysteine and serine tRNAs for seryl-tRNA synthetase is being determined. A total of 22 individual mutations has been made within the acceptor stem and in the extra loop (common sites of homology) in the corresponding tRNA genes. The ability of the products of these mutant forms to be aminoacylated by seryl-tRNA synthetase is presently being examined.
