Protein phosphorylation affects binding of the Escherichia coli transcription activator UhpA to the uhpT promoter
Protein phosphorylation affects binding of the Escherichia coli transcription activator UhpA to the uhpT promoter
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DOI：
10.1074/jbc.272.3.1910
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发表时间：
1997-01-17
影响因子：
4.8
通讯作者：
Kadner, RJ
中科院分区：
文献类型：
--
作者：
Dahl, JL;Wei, BY;Kadner, RJ
大肠杆菌糖磷酸转运蛋白UhpT的表达由胞外葡萄糖B-磷酸通过依赖于传感激酶UhpB和UhpT同源物UhpC的跨膜信号传导过程诱导。这些蛋白质被认为调节转录激活因子UhpA的磷酸化。为了检测蛋白磷酸化对UhpA与uhpT启动子区域中的靶序列结合的影响，过表达并纯化UhpA蛋白。纯化的UhpA磷酸化乙酰磷酸在反应中，这是依赖于镁离子和天冬氨酸54，在同源反应调节磷酸化的网站上的存在。凝胶电泳迁移率变化和DNA酶I和羟基自由基保护试验表明，UhpA特异性结合uhpT启动子的区域，从-80到-50 bp，相对于转录起始位点。在较高浓度的UhpA下，结合延伸至-32区域。结合到-64元件表现出积极的协同性，并刺激几倍的磷酸化UhpA，而延伸到下游区域的磷酸化的影响更强烈。提出了-64元件中高亲和力UhpA结合位点和下游低亲和力位点的共有序列。UhpA的体外结合模式与体内观察结果一致，即转录起始复合物的磷酸化独立组装可以在UhpA浓度升高时发生。
Expression of the Escherichia coli sugar phosphate transporter UhpT is induced by extracellular glucose B-phosphate through a transmembrane signaling process dependent on the sensor kinase UhpB and the UhpT homolog, UhpC. These proteins are thought to regulate the phosphorylation of the transcription activator, UhpA. To examine the effect of protein phosphorylation on the binding of UhpA to target sequences in the uhpT promoter region, the UhpA protein was overexpressed and purified. Purified UhpA was phosphorylated by acetyl phosphate in a reaction that was dependent on Mg2+ and on the presence of aspartate 54, the site of phosphorylation in homologous response regulators. Gel electrophoretic mobility shift and DNase I and hydroxyl radical protection assays showed that UhpA bound specifically to the region of the uhpT promoter extending from -80 to -50 bp, relative to the transcription start site. At higher concentrations of UhpA, binding was extended to the -32 region. Binding to the -64 element exhibited positive cooperativity and was stimulated severalfold by phosphorylation of UhpA, whereas extension to the downstream region was more strongly affected by phosphorylation. The consensus sequences for the high affinity UhpA-binding sites in the -64 element and for the downstream, low affinity sites are proposed. The pattern of in vitro binding by UhpA agreed with the in vivo observations that phosphorylation-independent assembly of the transcription initiation complex can occur at elevated concentrations of UhpA.