RECONSTITUTION OF THE BIOSYNTHETIC-PATHWAY OF SELENOCYSTEINE TRANSFER-RNAS IN XENOPUS-OOCYTES

Selenocysteine is cotranslationally introduced into a growing polypept ide in response to certain UGA codons in selenoprotein mRNAs. The bios ynthesis of this amino acid initiates by aminoacylation of specific tR NAs (designated tRNA[Se,]Sec) With serine and subsequent conversion of the serine moiety to selenocysteine. The resulting selenocysteyl-tRNA then donates selenocysteine to protein. In most higher vertebrate cel ls and tissues examined, multiple selenocysteine isoacceptors have bee n described. Two of these have been determined to differ by only a sin gle modified residue in the wobble position of the anticodon. In addit ion, the steady-state levels and relative distributions of these isoac ceptors have been shown to be influenced by the presence of selenium. In order to gain a better understanding of the relationship between th ese tRNAs and how they are regulated, both the Xenopus selenocysteine tRNA gene and an in vitro synthesized RNA have each been injected into Xenopus oocytes and their maturation analyzed. In this system, seleni um enhanced RNA stability and altered the distribution of isoacceptors that differ by a single ribose methylation. Interestingly, the biosyn thesis of one of these modified nucleosides (5-methylcar-boxymethyl-2' -O-methyluridine), which has been identified only in the wobble positi on of selenocysteine tRNA, also occurs in oocytes. Examination of the modified residues in both the naturally occurring Xenopus selenocytein e tRNA and the products generated from exogenous templates in oocytes demonstrated the faithful reconstruction of the biosynthetic pathway f or these tRNAs.