Regulation of high affinity nickel uptake in bacteria -: Ni2+-dependent interaction of NikR with wild-type and mutant operator sites
Regulation of high affinity nickel uptake in bacteria -: Ni2+-dependent interaction of NikR with wild-type and mutant operator sites
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DOI：
10.1074/jbc.m002232200
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发表时间：
2000-06-30
影响因子：
4.8
通讯作者：
Sauer, RT
中科院分区：
文献类型：
--
作者：
Chivers, PT;Sauer, RT
大肠杆菌通过ATP依赖的NikABCDE通透酶主动输入镍。NikR是转录因子的带-螺旋-螺旋家族的一种蛋白质，在存在过量浓度的细胞内镍的情况下抑制nikABCDE操纵子的表达。在此，通过足迹法和突变分析在nikABCDE启动子内鉴定NikR操作位点。该操纵子由两个二联体对称的5 '-GTATGA-3'识别序列组成，它们被16个碱基对隔开。GTATGA序列中的突变在体外降低NikR结合亲和力，并在体内降低P-nik-lacZ融合的抑制。此外，NikR被证明是镍离子的直接传感器。强的操纵子结合需要20-50 μ M镍的持续存在，这表明存在低亲和力镍结合位点，并且NikR二聚体还含有两个高亲和力镍结合位点。除了GTATGA位点和镍之外，高亲和力操纵子结合还需要NikR的C-末端结构域。
Escherichia coli actively imports nickel via the ATP-dependent NikABCDE permease. NikR, a protein of the ribbon-helix-helix family of transcription factors, represses expression of the nikABCDE operon in the presence of excessive concentrations of intracellular nickel. Here, the NikR operator site is identified within the nikABCDE promoter by footprinting and mutational analyses. The operator consists of two dyad-symmetric 5'-GTATGA-3' recognition sequences separated by 16 base pairs. Mutations in the GTATGA sequences reduce NikR binding affinity in vitro and reduce repression of a P-nik-lacZ fusion in vivo. Moreover, NikR is shown to be a direct sensor of nickel ions. Strong operator binding requires the continual presence of 20-50 mu M nickel, indicating the presence of a low affinity nickel-binding site, and NikR dimers also contain two high affinity nickel-binding sites. In addition to both GTATGA sites and nickel, high affinity operator binding also requires the C-terminal domain of NikR.