UTILIZATION OF SELENOCYSTEYL-TRNA([SER]SEC) AND SERYL-TRNA([SER]SEC) IN PROTEIN-SYNTHESIS

The UGA selenocysteine (Sec) codon in glutathione peroxidase mRNA and in selenoprotein P and the UGA stop codon in rabbit beta-globin mRNA w ere employed to study the utilization of Sec-tRNA([Ser]Sec) and Ser-tR NA([Ser]Sec) in protein synthesis. In vitro Ser-tRNA([Sec]Sec) served as a suppressor of the UGA Sec codon as well as the UGA stop codon, wh ile Sec-tRNA([Ser]Sec) did not. However, in vivo Sec-tRNA([Ser]Sec) di d donate Sec to glutathione peroxidase in Xenopus oocytes microinjecte d with glutathione peroxidase mRNA and Sec-tRNA A ribosome binding ass ay was devised to investigate the interaction of aminoacyl-tRNA, rabbi t reticulocyte ribosomes, and eukaryotic elongation factor 1 (eEF-1) i n response to the appropriate trinucleoside diphosphate template. Ser tRNA([Sec]Sec) bound weakly to ribosomes in the presence of eEF-1 and UGA as compared to Phe-tRNA, Ser-tRNA(IGA) and Met-tRNA(m) which bound more efficiently in the presence of eEF-1 and the appropriate templat e. No increase in the binding of Sec-tRNA([Ser]Sec) was observed under the same conditions as Ser-tRNA([Ser]Sec.) The ribosome binding studi es substantiated the finding that Ser-tRNA([Ser]Sec) serves as a suppr essor of UGA codons in protein synthesis, but SectRNA([Ser]Sec) does n ot. In addition, these studies provide strong evidence that a specific elongation factor is required in mammalian cells for insertion of Sec into protein from Sec-tRNA([Ser]Sec).