Mettke I, Fiedler U, Weiss V
Department of Biology, University of Konstanz, Germany.
J Bacteriol. 1995 Sep;177(17):5056-61. doi: 10.1128/jb.177.17.5056-5061.1995.
NtrC is the transcriptional activator for nitrogen-regulated promoters and, as a response regulator, belongs to the protein family of two-component systems. The activity of all response regulators is modulated by phosphorylation of the conserved N-terminal receiver domain. Phosphorylation of the dimeric NtrC has two consequences: (i) a strong increase in the cooperative binding of NtrC to two adjacent binding sites and (ii) activation of NtrC as an ATPase. Here we show that phosphorylation of NtrC is not sufficient for activation of NtrC. At low protein concentrations (50 nM), phosphorylated NtrC was only active as an ATPase upon cooperative binding to DNA. At high protein concentrations (above 50 nM), NtrC was active in the absence of DNA, and activation occurred in parallel with the formation of high-molecular-weight aggregates. We infer that activation of NtrC involves an interaction between two NtrC-P dimers and proceeds in two steps. The first step is the phosphorylation of NtrC. The second step is the interaction between two NtrC-P dimers. This interaction induces the conformational change in NtrC-P to the active conformation.
NtrC是氮调节启动子的转录激活因子，作为一种应答调节因子，属于双组分系统的蛋白质家族。所有应答调节因子的活性都通过保守的N端接收结构域的磷酸化来调节。二聚体NtrC的磷酸化有两个结果：（i）NtrC与两个相邻结合位点的协同结合显著增加；（ii）NtrC作为ATP酶被激活。在这里，我们表明NtrC的磷酸化不足以激活NtrC。在低蛋白浓度（50 nM）下，磷酸化的NtrC只有在与DNA协同结合时才作为ATP酶具有活性。在高蛋白浓度（高于50 nM）下，NtrC在没有DNA的情况下具有活性，并且激活与高分子量聚集体的形成同时发生。我们推断，NtrC的激活涉及两个NtrC-P二聚体之间的相互作用，并分两步进行。第一步是NtrC的磷酸化。第二步是两个NtrC-P二聚体之间的相互作用。这种相互作用诱导NtrC-P的构象变化为活性构象。
