Michán C M, Busby S J, Hyde E I
School of Biochemistry, University of Birmingham, UK.
Nucleic Acids Res. 1995 May 11;23(9):1518-23. doi: 10.1093/nar/23.9.1518.
A set of nested deletions has been made in the Escherichia coli melR gene, encoding the MelR transcription activator protein. Expression of the resulting melR derivatives led to the production of nine MelR proteins with N-terminal deletions of different lengths. The properties of the shortened proteins have been studied both in vivo and in vitro. None of the truncated proteins activate transcription from the E.coli melAB promoter but three; MelR220, MelR183 and MelR173, inhibit activation of the melAB promoter by chromosomally-encoded full-length MelR. In gel retardation assays, both MelR183 and MelR173 clearly retard DNA fragments carrying the melAB promoter. MelR173 has been overproduced in a T7 expression system and shown to be stable in vivo for up to 2 h. DNAase I footprinting assays of partially purified protein show that it binds to the melAB promoter, protecting the same sites as the full-length protein. This fragment may be suitable for further structure/function studies of this class of transcription activator.
已在编码MelR转录激活蛋白的大肠杆菌melR基因中进行了一系列嵌套缺失。所得melR衍生物的表达导致产生了九种具有不同长度N端缺失的MelR蛋白。已在体内和体外研究了这些缩短蛋白的特性。截短的蛋白中没有一种能激活大肠杆菌melAB启动子的转录，但MelR220、MelR183和MelR173这三种蛋白能抑制染色体编码的全长MelR对melAB启动子的激活。在凝胶阻滞试验中，MelR183和MelR173都能明显阻滞携带melAB启动子的DNA片段。MelR173已在T7表达系统中过量表达，并显示在体内长达2小时内稳定。对部分纯化蛋白的DNA酶I足迹分析表明，它与melAB启动子结合，保护与全长蛋白相同的位点。该片段可能适用于此类转录激活剂的进一步结构/功能研究。
