Lahue E E, Matson S W
Department of Biology and Curriculum in Genetics, University of North Carolina, Chapel Hill 27599.
J Bacteriol. 1990 Mar;172(3):1385-91. doi: 10.1128/jb.172.3.1385-1391.1990.
The traY gene of the Escherichia coli F plasmid has been shown by genetic studies (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980) to be involved in the site-specific nicking reaction at oriT required for the initiation of DNA transfer during bacterial conjugation. In order to assign a biochemical function to TraY protein, the traY gene was cloned in a plasmid vector which utilizes the strong T7 phi 10 promoter to overproduce the protein. The plasmid-encoded TraY protein was specifically labeled with [35S]methionine, and purification of the polypeptide was accomplished by monitoring the radioactive label. Purified TraY protein had a relative molecular mass of approximately 17,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino terminus of the purified protein was sequenced to confirm that the protein was encoded by the traY gene. The protein sequence revealed that the start codon for the TraY protein was a UUG codon 36 base pairs upstream of the AUG start site originally deduced from the DNA sequence (T. Fowler, L. Taylor, and R. Thompson, Gene 26:79-89, 1983). This start sequence confirmed the premise of Inamoto et al. that the F-plasmid TraY polypeptide-coding sequence would begin with UUG, creating a reading frame which renders a large degree of amino acid sequence identity with the TraY polypeptide from R100 (S. Inamoto, Y. Yoshioka, and E. Ohtsubo, J. Bacteriol. 170:2749-2757, 1988). The purified TraY protein from F bound specifically to the origin of transfer region of the F plasmid. However, no nicking activity was detected at oriT by using TraY protein or TraY protein in conjunction with helicase I.
遗传学研究（R. 埃弗雷特和N. 威利茨，《分子生物学杂志》136:129 - 150，1980年）表明，大肠杆菌F质粒的traY基因参与细菌接合过程中DNA转移起始所需的oriT位点特异性切口反应。为了赋予TraY蛋白生化功能，将traY基因克隆到一个质粒载体中，该载体利用强T7 phi 10启动子过量表达该蛋白。用[35S]甲硫氨酸对质粒编码的TraY蛋白进行特异性标记，并通过监测放射性标记来完成多肽的纯化。经十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳测定，纯化的TraY蛋白相对分子质量约为17,000。对纯化蛋白的氨基末端进行测序，以确认该蛋白由traY基因编码。蛋白质序列显示，TraY蛋白的起始密码子是一个UUG密码子，位于最初从DNA序列推导的AUG起始位点上游36个碱基对处（T. 福勒、L. 泰勒和R. 汤普森，《基因》26:79 - 89，1983年）。这个起始序列证实了稻本等人的假设，即F质粒TraY多肽编码序列将以UUG开头，形成一个阅读框，使其与R100的TraY多肽具有高度的氨基酸序列同一性（S. 稻本、吉冈洋和大坪悦郎，《细菌学杂志》170:2749 - 2757，1988年）。从F质粒纯化的TraY蛋白特异性结合到F质粒的转移起始区域。然而，使用TraY蛋白或TraY蛋白与解旋酶I结合时，在oriT处未检测到切口活性。
