A putative copper ion-sensing transcriptional regulator CopR (TON_0836) from Thermococcus onnurineus NA1 was characterized. The CopR protein consists of a winged helix-turn-helix DNA-binding domain in the amino-terminal region and a TRASH domain that is assumed to be involved in metal ion-sensing in the carboxyl-terminal region. The CopR protein was most strongly bound to a region between its own gene promoter and a counter directional promoter region for copper efflux system CopA. When the divalent metals such as nickel, cobalt, copper, and iron were present, the CopR protein was dissociated from the target promoters on electrophoretic mobility shift assay (EMSA). The highest sensible ion is copper which affected protein releasing under 10 µM concentrations. CopR recognizes a significant upstream region of TATA box on CopR own promoter and acts as a transcriptional repressor in an in vitro transcription assay. Through site-directed mutagenesis of the DNA-binding domain, R34M mutant protein completely lost the DNA-binding activity on target promoter. When the conserved cysteine residues in C144XXC147 motif 1 of the TRASH domain were mutated into glycine, the double cysteine residue mutant protein alone lost the copper-binding activity. Therefore, CopR is a copper-sensing transcriptional regulator and acts as a repressor for autoregulation and for a putative copper efflux system CopA of T. onnurineus NA1.

中文翻译：

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嗜铜古菌Thermococcus onnurineus NA1的铜敏感转录调节因子CopR的表征。

推定的铜离子感测转录调节因子CopR（TON_0836）来自Thermococcus onnurineus NA1。CopR蛋白由氨基末端区域的带翼螺旋-转-螺旋-DNA结合结构域和TRASH结构域组成，TRASH结构域被认为与羧基末端区域的金属离子感测有关。CopR蛋白与铜外排系统CopA的基因启动子和反向启动子区域之间的结合最牢固。当存在二价金属（例如镍，钴，铜和铁）时，在电泳迁移率迁移分析（EMSA）上CopR蛋白从靶标启动子上解离。最高的敏感离子是铜，它会影响10 µM浓度下的蛋白质释放。CopR识别CopR自身启动子上TATA盒的重要上游区域，并在体外转录测定中充当转录阻遏物。通过DNA结合域的定点诱变，R34M突变蛋白完全失去了对靶启动子的DNA结合活性。当TRASH域的C144XXC147基序1中的保守半胱氨酸残基突变为甘氨酸时，双半胱氨酸残基突变蛋白单独失去了铜结合活性。因此，CopR是一种铜敏感的转录调节因子，可作为自动调节因子和on.nunurineus NA1的假定铜外排系统CopA的阻遏物。R34M突变蛋白完全失去了对靶启动子的DNA结合活性。当TRASH域的C144XXC147基序1中的保守半胱氨酸残基突变为甘氨酸时，双半胱氨酸残基突变蛋白单独失去了铜结合活性。因此，CopR是一种铜敏感的转录调节因子，并充当自动调节和on T. onnurineus NA1的假定铜外排系统CopA的阻遏物。R34M突变蛋白完全失去了对靶启动子的DNA结合活性。当TRASH域的C144XXC147基序1中的保守半胱氨酸残基突变为甘氨酸时，双半胱氨酸残基突变蛋白单独失去了铜结合活性。因此，CopR是一种铜敏感的转录调节因子，可作为自动调节因子和on.nunurineus NA1的假定铜外排系统CopA的阻遏物。