Jung J E, Karoor V, Sandbaken M G, Lee B J, Ohama T, Gesteland R F, Atkins J F, Mullenbach G T, Hill K E, Wahba A J
Laboratory of Experimental Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Nov 25;269(47):29739-45.
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.
利用谷胱甘肽过氧化物酶mRNA、硒蛋白P中的UGA硒代半胱氨酸（Sec）密码子以及兔β-珠蛋白mRNA中的UGA终止密码子，研究了Sec-tRNA[Ser]Sec和Ser-tRNA[Ser]Sec在蛋白质合成中的利用情况。在体外，Ser-tRNA[Ser]Sec可作为UGA Sec密码子以及UGA终止密码子的抑制子，而Sec-tRNA[Ser]Sec则不能。然而，在体内，将谷胱甘肽过氧化物酶mRNA和Sec-tRNA显微注射到非洲爪蟾卵母细胞后，Sec-tRNA[Ser]Sec确实将Sec提供给了谷胱甘肽过氧化物酶。设计了一种核糖体结合试验，以研究氨酰-tRNA、兔网织红细胞核糖体和真核延伸因子1（eEF-1）在响应适当的三磷酸核苷模板时的相互作用。与在eEF-1和适当模板存在下结合更有效的苯丙氨酰-tRNA、丝氨酰-tRNAIGA和甲硫氨酰-tRNA相比，在eEF-1和UGA存在的情况下，Ser-tRNA[Ser]Sec与核糖体的结合较弱。在与Ser-tRNA[Ser]Sec相同的条件下，未观察到Sec-tRNA[Ser]Sec结合的增加。核糖体结合研究证实了Ser-tRNA[Ser]Sec在蛋白质合成中作为UGA密码子抑制子的发现，但Sec-tRNA[Ser]Sec则不是。此外，这些研究提供了强有力的证据，表明哺乳动物细胞中需要一种特定的延伸因子才能将Sec从Sec-tRNA[Ser]Sec插入蛋白质中。
