Olekhnovich I N, Dahl J L, Kadner R J
Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
J Mol Biol. 1999 Oct 8;292(5):973-86. doi: 10.1006/jmbi.1999.3127.
Activation of promoters by multiple transcription factors might occur through favorable contacts of the activators with themselves or RNA polymerase, or by changes in DNA geometry that enhance formation of the transcription complex. Transcription of the Escherichia coli uhpT gene, encoding the organophosphate transporter, requires the response regulator UhpA and is stimulated by the global regulator protein CAP. CAP binds to the uhpT promoter at a single site, centered at -103.5 bp relative to the start of transcription, and UhpA binds to multiple sites between positions -80 and -32. Overexpression of UhpA did not reduce the degree of CAP stimulation of uhpT-lacZ expression, showing that CAP action is more complex than enhancement of the binding of UhpA. Footprinting experiments demonstrated that UhpA and CAP modestly stimulated each other's binding to the uhpT promoter, but did not affect the positioning of the binding sites. An in vitro transcription system was used to examine the contribution of each transcription factor at the uhpT promoter. Action of UhpA and CAP proteins was not affected by template supercoiling. Kinetic analyses of productive and abortive initiation showed that CAP acted both to stabilize by fivefold the open promoter complexes formed in the presence of UhpA and to enhance by twofold the rate of their formation. These results indicate that open complex formation requires UhpA and that CAP stabilizes the open complex.
多个转录因子对启动子的激活可能是通过激活因子自身之间或与RNA聚合酶的有利接触，或者是通过增强转录复合物形成的DNA几何结构变化来实现的。编码有机磷酸转运蛋白的大肠杆菌uhpT基因的转录需要应答调节因子UhpA，并受到全局调节蛋白CAP的刺激。CAP在一个位点结合到uhpT启动子上，该位点相对于转录起始点位于-103.5 bp处，而UhpA在-80至-32位之间的多个位点结合。UhpA的过表达并没有降低CAP对uhpT-lacZ表达的刺激程度，这表明CAP的作用比增强UhpA的结合更为复杂。足迹实验表明，UhpA和CAP适度地相互刺激对方与uhpT启动子的结合，但不影响结合位点的定位。使用体外转录系统来检测每个转录因子在uhpT启动子上的作用。UhpA和CAP蛋白的作用不受模板超螺旋的影响。对有效起始和无效起始的动力学分析表明，CAP既使在存在UhpA的情况下形成的开放启动子复合物稳定了五倍，又使它们的形成速率提高了两倍。这些结果表明开放复合物的形成需要UhpA，而CAP则稳定开放复合物。
