Utilization of selenocysteyl-tRNA super([Ser]Sec) and seryl-tRNA super([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 were employed to study the utilization of Sec-tRNA super([Ser]Sec) and Ser-tRNA super([Ser]Sec) in protein synthesis. In vitro Ser-tRNA super([Ser]Sec) served as a suppressor of the UGA Sec codon as well as the UGA stop codon, while Sec-tRNA super([Ser]Sec) did not. However, in vivo Sec-tRNA super([Ser]Sec) did donate Sec to glutathione peroxidase in Xenopus oocytes microinjected with glutathione peroxidase mRNA and Sec-tRNA. A ribosome binding assay was devised to investigate the interaction of aminoacyl-tRNA, rabbit reticulocyte ribosomes, and eukaryotic elongation factor 1 (eEF-1) in response to the appropriate trinucleoside diphosphate template. Ser-tRNA super([Ser]Sec) bound weakly to ribosomes in the presence of eEF-1 and UGA as compared to Phe-tRNA, Ser-tRNA sub(IGA), and Met-tRNA super(m) which bound more efficiently in the presence of eEF-1 and the appropriate template. No increase in the binding of Sec-tRNA super([Ser]Sec) was observed under the same conditions as Ser-tRNA super([Ser]Sec). The ribosome binding studies substantiated the finding that Ser-tRNA super([Ser]Sec) serves as a suppressor of UGA codons in protein synthesis, but Sec-tRNA super([Ser]Sec) does not. In addition, the studies provide strong evidence that a specific elongation factor is required in mammalian cells for insertion of Sec into protein from Sec-tRNA super([Ser]Sec).