Liu W, Qi Y, Hulett F M
Department of Biological Sciences, University of Illinois at Chicago, 60607, USA.
Mol Microbiol. 1998 Apr;28(1):119-30. doi: 10.1046/j.1365-2958.1998.00779.x.
Bacillus subtilis PhoP and PhoR, a pair of two-component regulatory proteins, regulate the phosphate starvation response. Here, we used two other pho regulon promoters, the phoA and pstS promoters, to examine the mechanism of PhoP-specific activation of its target promoters. Both gel shift and DNase I footprinting assays indicate that PhoP bound to the two promoters. Unphosphorylated PhoP bound only to the multiple TTAACA-like sequences upstream of these two promoters, while phosphorylated PhoP extended the binding region in both the 5' and the 3' direction and, additionally, protected sequences internal to the coding region of these two genes. The PhoP binding sites in the coding region were necessary for full induction from either promoter during phosphate starvation. Deletion of these sites eliminated approximately 75% and 45% of the induced promoter activity of the phoA and pstS promoters respectively. In vitro transcription assays using the phoA promoters with various 3' ends confirmed the requirement of the PhoP-P binding to the coding region for full promoter activity. The multiple TTAACA-like sequences in the phoA and pstS promoters were essential for promoter activity, and deletion of one or more of these sequences in either promoter eliminated the promoter activity. Two pairs of TTAACA-like sequences were required for efficient PhoP binding and were suggested to be one B. subtilis Pho box. Based on our data, we have proposed a model for activation of the phoA and the pstS promoter by PhoP.
枯草芽孢杆菌的PhoP和PhoR是一对双组分调节蛋白，它们调节磷酸盐饥饿反应。在此，我们使用另外两个pho调控子启动子，即phoA和pstS启动子，来研究PhoP对其靶启动子的特异性激活机制。凝胶迁移和DNase I足迹分析均表明PhoP与这两个启动子结合。未磷酸化的PhoP仅与这两个启动子上游的多个TTAACA样序列结合，而磷酸化的PhoP在5'和3'方向上均扩展了结合区域，此外，还保护了这两个基因编码区域内的序列。在磷酸盐饥饿期间，编码区域中的PhoP结合位点对于从任一启动子进行完全诱导都是必需的。缺失这些位点分别消除了phoA和pstS启动子约75%和45%的诱导启动子活性。使用具有不同3'末端的phoA启动子进行的体外转录分析证实了PhoP-P与编码区域结合对于启动子完全活性的必要性。phoA和pstS启动子中的多个TTAACA样序列对于启动子活性至关重要，在任一启动子中缺失一个或多个这些序列都会消除启动子活性。两对TTAACA样序列是PhoP有效结合所必需的，并且被认为是一个枯草芽孢杆菌Pho框。基于我们的数据，我们提出了一个PhoP激活phoA和pstS启动子的模型。
