Chen Q, Kadner R J
Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0734, USA.
J Bacteriol. 2000 Aug;182(16):4430-6. doi: 10.1128/JB.182.16.4430-4436.2000.
Many bacterial promoters possess multiple sites for binding of transcriptional activator proteins. The uhpT promoter, which controls expression of the sugar phosphate transport system in Escherichia coli, possesses multiple sites for its specific activator protein, UhpA, and a single site for binding of the global regulator, the catabolite gene activator protein (CAP). The binding of UhpA to the uhpT promoter was determined by DNase protection assays; UhpA displayed different affinities for the target sites. The upstream or strong sites, between positions -80 and -50, exhibited a higher affinity for UhpA than did the downstream or weak sites, between positions -50 and -32, adjoining the RNA polymerase-binding site. Phosphorylation of UhpA strongly increased its affinity for both sites. To examine the possible roles of the two sets of UhpA-binding sites, a series of insertion and deletion mutations were introduced at the boundary between them, as suggested from the positions that were protected by UhpA against hydroxyl radical cleavage. Deletions extended in the direction of the weak sites. The insertion or deletion of one helical turn of DNA resulted in the loss of promoter activity and of occupancy by UhpA of the remaining weak-site sequences but was accompanied by normal occupancy of the strong site and no change in the gel retardation behavior of the promoter fragments. However, the deletion of two helical turns of DNA, i.e., 20, 21, or 22 bp, resulted in the novel appearance of UhpA-independent expression and in an additional level of expression that was dependent on UhpA but independent of an inducing signal. The UhpA-independent promoter activity was shown to result from activation by CAP at its more proximal position. UhpA-dependent activity under noninducing conditions appears to result from the binding of unphosphorylated UhpA to the strong sites, which are now in the position normally occupied by the weak sites. Thus, regulated phosphorylation of the response regulator UhpA enhances its occupancy of the weak sites where favorable contacts can allow the binding of RNA polymerase to the promoter.
许多细菌启动子具有多个转录激活蛋白结合位点。控制大肠杆菌中磷酸糖转运系统表达的uhpT启动子，具有多个其特异性激活蛋白UhpA的结合位点，以及一个用于结合全局调节因子——分解代谢基因激活蛋白（CAP）的单一位点。通过DNA酶保护试验确定了UhpA与uhpT启动子的结合；UhpA对靶位点表现出不同的亲和力。位于-80至-50位之间的上游或强位点对UhpA的亲和力高于位于-50至-32位之间、毗邻RNA聚合酶结合位点的下游或弱位点。UhpA的磷酸化强烈增加了其对这两个位点的亲和力。为了研究两组UhpA结合位点的可能作用，根据UhpA保护免受羟基自由基切割的位置提示，在它们之间的边界处引入了一系列插入和缺失突变。缺失向弱位点方向延伸。DNA一螺旋圈的插入或缺失导致启动子活性丧失以及UhpA对剩余弱位点序列的占据丧失，但同时强位点的正常占据以及启动子片段的凝胶阻滞行为没有变化。然而，DNA两螺旋圈（即20、21或22 bp）的缺失导致了不依赖UhpA的表达的新出现，以及依赖UhpA但不依赖诱导信号的额外表达水平。已证明不依赖UhpA 的启动子活性是由CAP在其更近端位置的激活导致的。非诱导条件下依赖UhpA的活性似乎是由于未磷酸化的UhpA与强位点的结合，而这些强位点现在处于通常由弱位点占据的位置。因此，应答调节因子UhpA的磷酸化调节增强了其对弱位点的占据，在这些弱位点上有利的接触可以使RNA聚合酶与启动子结合。
