Metal-selective DNA-binding response of Escherichia coli NikR
Metal-selective DNA-binding response of Escherichia coli NikR
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DOI：
10.1021/bi049404k
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发表时间：
2004-08-10
期刊：
影响因子：
2.9
通讯作者：
Zamble, DB
中科院分区：
文献类型：
--
作者：
Bloom, SL;Zamble, DB
来自大肠杆菌的NikR转录因子是Ni（II）依赖性阻遏物，其调节由nik操纵子编码的镍离子转运蛋白的产生。在本期的前一篇论文中（Wang，S。C.的方法，迪亚斯，A.，Bloom，S. L.，和Zamble，D. B。（2004）Selectivity of Metal Binding and Metal-Induced Stability of Escherichia coli NikR，Biochemistry 43，10018-10028），我们证明了NikR可以结合1当量的Ni（II）或具有类似亲和力的几种其他二价过渡金属，但是负载Ni（II）的蛋白质比其他二价金属络合物更不易于热或化学变性。在这里，我们调查的金属选择性的DNA结合活性的NikR。化学计量的镍诱导纳摩尔的NikR与nik启动子中的识别序列的结合，但单一当量的其他二价金属如Cd（II）、Co（II）和Cu（II）也诱导类似的DNA结合亲和力。在过量镍的存在下，DNA结合实验表明NikR以高得多的亲和力（20 pM）作为四聚体与nik启动子结合，并且正是这种响应对镍具有选择性。由过量的其他二价金属诱导的DNA结合较弱，并且通过添加化学计量的镍而增强。DNA结合试验中的镍滴定显示镍对第二金属结合位点的亲和力为30 nM，并且在存在30 nM金属的情况下，仅镍诱导Ni（II）-NikR可检测到的DNA结合。这些实验支持的假设，有两个金属结合位点，都有助于镍选择性DNA结合反应。讨论了NIKR体内活性的模型。
The NikR transcription factor from Escherichia coli is a Ni(II)-dependent repressor that regulates production of the nickel ion transporter encoded by the nik operon. In the previous paper in this issue (Wang, S. C., Dias, A., Bloom, S. L., and Zamble, D. B. (2004) Selectivity of Metal Binding and Metal-Induced Stability of Escherichia coli NikR, Biochemistry 43, 10018-10028) we demonstrated that NikR can bind 1 equiv of Ni(11) or several other divalent transition metals with similar affinities, but that the Ni(II)-loaded protein is less susceptible to thermal or chemical denaturation than other divalent metal complexes. Here, we investigate the metal selectivity of the DNA-binding activity of NikR. Stoichiometric nickel induces binding of nanomolar NikR to the recognition sequence in the nik promoter, but single equivalents of other divalent metals such as Cd(II), Co(II), and Cu(II) also induce a similar DNA-binding affinity. In the presence of excess nickel, DNA-binding experiments indicate that NikR binds to the nik promoter as a tetramer with much higher affinity (20 pM), and it is this response that is selective for nickel. The DNA binding induced by an excess of other divalent metals is weaker, and is enhanced by the addition of stoichiometric nickel. Nickel titrations into a DNA-binding assay reveal a nickel affinity of 30 nM for a second metal-binding site, and in the presence of 30 nM metal only nickel induces detectable DNA binding by Ni(II)-NikR. These experiments support the hypothesis that there are two metal-binding sites and that both contribute to the nickel-selective DNA-binding response. A model for the in vivo activity of N ikR is discussed.