Wen W, Weiss S L, Sunde R A
Nutritional Sciences Program, University of Missouri, Columbia, Missouri 65211, USA.
J Biol Chem. 1998 Oct 23;273(43):28533-41. doi: 10.1074/jbc.273.43.28533.
A UGA codon and a selenocysteine insertion sequence in the 3'-untranslated region are the only established mRNA elements necessary for selenocysteine (Sec or U) incorporation during translation. These two elements, however, do not universally confer efficient Sec incorporation. The objective of this study was to systematically examine the effect of UGA codon position on efficiency of Sec insertion. In a glutathione peroxidase-1 (F-GPX1) expression vector, the UGA at the native position (U47) was mutated to a cysteine codon, and codons for Ser-7, Ser-12, Ser-18, Ser-29, Ser-45, Ser-93, Cys-154, Val-172, Ser-178, and Ser-195 were individually mutated to UGA and transiently expressed in COS-7 cells. 75Se incorporation at the 11 positions was 31, 72, 54, 105, 90, 100, 146, 135, 13, 11, and 43%, respectively, of 75Se incorporation at U47, suggesting that Sec is more efficiently incorporated at UGA codons positioned in the middle of the coding region rather than close to the 5' or 3' ends. Ribonuclease protection showed that these differences were not due to differences in mRNA level. When the green fluorescence protein (GFP) coding region was placed in-frame at the 5' or 3' ends of the coding region in F-GPX1 to produce chimeric 50-51-kDa GFP/GPX1 proteins, Sec incorporation at UGA codons, formerly close to the 5' or 3' ends, was increased to levels comparable to the UGA at U47. Insertion of GFP after the UAA-stop was just as effective in increasing Sec insertion efficiency as GFP inserted before the stop. These studies used a recombinant expression model that incorporated Sec at non-native UGA codons at rates equal to those of endogenous glutathione peroxidase-1 and showed that the efficiency of Sec incorporation can be modulated by UGA position; Sec incorporation at high efficiency appears to require that the UGA be >21 nucleotides from the AUG-start and >204 nucleotides from the selenocysteine insertion sequence element.
3'-非翻译区中的UGA密码子和硒代半胱氨酸插入序列是翻译过程中硒代半胱氨酸（Sec或U）掺入所必需的仅有的已确定的mRNA元件。然而，这两个元件并不能普遍赋予高效的Sec掺入。本研究的目的是系统地研究UGA密码子位置对Sec插入效率的影响。在谷胱甘肽过氧化物酶-1（F-GPX1）表达载体中，将天然位置（U47）的UGA突变为半胱氨酸密码子，并将Ser-7、Ser-12、Ser-18、Ser-29、Ser-45、Ser-93、Cys-154、Val-172、Ser-178和Ser-195的密码子分别突变为UGA，并在COS-7细胞中瞬时表达。这11个位置的75Se掺入量分别为U47处75Se掺入量的31%、72%、54%、105%、90%、100%、146%、135%、13%、11%和43%，这表明Sec在位于编码区中间的UGA密码子处比在靠近5'或3'端处更有效地掺入。核糖核酸酶保护显示这些差异不是由于mRNA水平的差异。当绿色荧光蛋白（GFP）编码区框内置于F-GPX1编码区的5'或3'端以产生嵌合的50 - 51 kDa GFP/GPX1蛋白时，以前靠近5'或3'端的UGA密码子处的Sec掺入量增加到与U47处的UGA相当的水平。在UAA终止密码子之后插入GFP在提高Sec插入效率方面与在终止密码子之前插入GFP同样有效。这些研究使用了一种重组表达模型，该模型以与内源性谷胱甘肽过氧化物酶-1相同的速率在非天然UGA密码子处掺入Sec，并表明Sec掺入效率可由UGA位置调节；高效的Sec掺入似乎要求UGA距离AUG起始密码子>21个核苷酸且距离硒代半胱氨酸插入序列元件>204个核苷酸。
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