Zinoni F, Birkmann A, Stadtman T C, Böck A
Proc Natl Acad Sci U S A. 1986 Jul;83(13):4650-4. doi: 10.1073/pnas.83.13.4650.
The gene (fdhF) coding for the selenopolypeptide of the benzylviologen-linked formate dehydrogenase of Escherichia coli was cloned and its nucleotide sequence was determined. The fdhF gene contains, within an open reading frame coding for a protein of 715 amino acids (calculated molecular weight, 79,087), an opal (UGA) nonsense codon in amino acid position 140. Existence of this nonsense codon was confirmed by physical recloning and resequencing. Internal and terminal deletion clones and lacZ fusions of different N-terminal parts of fdhF were constructed and analyzed for selenium incorporation. Selenylated truncated polypeptide chains or beta-galactosidase fusion proteins were synthesized when the deletion clones or gene fusions, respectively, contained the fdhF gene fragment coding for the selenopolypeptide sequence from amino acid residue 129 to amino acid residue 268. Translation of the lacZ part of the fusions required the presence of selenium in the medium when the N-terminal fdhF part contained the UGA codon and was independent of the presence of selenium when a more upstream part of fdhF was fused to lacZ. The results are consistent with a co-translational selenocysteine incorporation mechanism.
编码大肠杆菌苄基紫精连接的甲酸脱氢酶硒代多肽的基因（fdhF）被克隆，并测定了其核苷酸序列。fdhF基因在一个编码715个氨基酸（计算分子量为79,087）的开放阅读框内，在氨基酸位置140处有一个乳白（UGA）无义密码子。通过物理重克隆和重新测序证实了这个无义密码子的存在。构建了fdhF不同N端部分的内部和末端缺失克隆以及lacZ融合体，并分析了硒的掺入情况。当缺失克隆或基因融合体分别包含编码从氨基酸残基129到氨基酸残基268的硒代多肽序列的fdhF基因片段时，合成了硒化的截短多肽链或β-半乳糖苷酶融合蛋白。当融合体的lacZ部分的翻译需要培养基中存在硒时，N端的fdhF部分包含UGA密码子；而当fdhF的更上游部分与lacZ融合时，翻译则与硒的存在无关。结果与共翻译硒代半胱氨酸掺入机制一致。
