Harrison-McMonagle P, Denissova N, Martínez-Hackert E, Ebright R H, Stock A M
Center for Advanced Biotechnology and Medicine, 679 Hoes Ln, Piscataway, NJ, 08854, USA.
J Mol Biol. 1999 Jan 15;285(2):555-66. doi: 10.1006/jmbi.1998.2375.
Escherichia coli OmpR is a transcription factor that regulates the differential expression of the porin genes ompF and ompC. Phosphorylated OmpR binds as a dimer to a 20-bp region of DNA consisting of two tandemly arranged 10-bp half-sites. Expression of the ompF gene is achieved by the hierarchical occupation of three adjacent 20-bp binding sites, designated F1, F2, and F3 and a distally located site, F4. Despite genetic, biochemical, and structural studies, specific details of the interaction between phosphorylated OmpR and the DNA remain unknown. We have linked the DNA cleaving moiety o-phenanthroline-copper to eight different sites within the DNA binding domain of OmpR in order to determine the orientation of the two OmpR monomers in the OmpR:F1 complex. Five of the resulting conjugates exhibited DNA cleaving activity, and four of these yielded patterns that could be used to construct a model of the OmpR:F1 complex. We propose that OmpR binds asymmetrically to the F1 site as a tandemly arranged dimer with each monomer having its recognition helix in the major groove. The N-terminal end of the recognition helix is promoter-proximal and flanked by "wings" W1 and W2 positioned proximally and distally, respectively, to the transcription start site of ompF. We further propose that the C-terminal end of the recognition helix makes the most extensive contacts with DNA and predict bases within the F1 site that are sufficiently close to be contacted by the recognition helix.
大肠杆菌OmpR是一种转录因子，可调节孔蛋白基因ompF和ompC的差异表达。磷酸化的OmpR以二聚体形式结合到由两个串联排列的10 bp半位点组成的20 bp DNA区域。ompF基因的表达是通过依次占据三个相邻的20 bp结合位点（分别命名为F1、F2和F3）以及一个位于远端的位点F4来实现的。尽管进行了遗传学、生物化学和结构研究，但磷酸化的OmpR与DNA之间相互作用的具体细节仍不清楚。我们将DNA切割部分邻菲罗啉 - 铜连接到OmpR DNA结合结构域内的八个不同位点，以确定OmpR：F1复合物中两个OmpR单体的方向。所得的五个缀合物表现出DNA切割活性，其中四个产生的模式可用于构建OmpR：F1复合物的模型。我们提出，OmpR作为串联排列的二聚体不对称地结合到F1位点，每个单体的识别螺旋位于大沟中。识别螺旋的N末端靠近启动子，两侧分别是位于ompF转录起始位点近端和远端的“翼”W1和W2。我们进一步提出，识别螺旋C末端与DNA的接触最为广泛，并预测F1位点内足够接近识别螺旋可接触的碱基。
