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 were employed to study the utilization of Sec-tRNA[Ser]Sec and Ser-tRNA[Ser]Sec in protein synthesis. In vitro Ser-tRNA[Ser]Sec served as a suppressor of the UGA Sec codon as well as the UGA stop codon, while Sec-tRNA[Ser]Sec did not. However, in vivo Sec-tRNA[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[Ser]Sec bound weakly to ribosomes in the presence of eEF-1 and UGA as compared to Phe-tRNA, Ser-tRNAIGA, and Met-tRNAm which bound more efficiently in the presence of eEF-1 and the appropriate template. No increase in the binding of Sec-tRNA[Ser]Sec was observed under the same conditions as Ser-tRNA[Ser]Sec. The ribosome binding studies substantiated the finding that Ser-tRNA[Ser]Sec serves as a suppressor of UGA codons in protein synthesis, but Sec-tRNA[Ser]Sec does not. 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.
Laboratory of Experimental Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland 20892.
J E
JE
Jung
V
V
Karoor
M G
MG
Sandbaken
B J
BJ
Lee
T
T
Ohama
R F
RF
Gesteland
J F
JF
Atkins
G T
GT
Mullenbach
K E
KE
Hill
A J
AJ
Wahba
D000215:Acylation; D000818:Animals; D003062:Codon; D005979:Glutathione Peroxidase / Q000378:metabolism; D009154:Mutation; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism
D003062:Codon; D012346:RNA, Transfer, Amino Acyl; C112705:selenocysteinyl-tRNA; D005979:Glutathione Peroxidase