Department of Biology, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, South Korea.
Educational Administration of Gyonggido, Euijungbu, 11759, South Korea.
Biometals. 2019 Dec;32(6):923-937. doi: 10.1007/s10534-019-00223-2. Epub 2019 Nov 1.
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 CXXC 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.
从嗜热球菌中鉴定出一种假定的铜离子感应转录调节因子 CopR（TON_0836）。CopR 蛋白由氨基末端区域的翅膀螺旋-转角-螺旋 DNA 结合域和假定参与羧基末端区域金属离子感应的 TRASH 结构域组成。CopR 蛋白与自身基因启动子和铜外排系统 CopA 的反向启动子区域之间的区域结合最强。在存在二价金属如镍、钴、铜和铁时，CopR 蛋白在电泳迁移率变动分析（EMSA）中从靶启动子解离。最敏感的离子是铜，在 10µM 浓度下影响蛋白释放。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作为转录抑制剂。通过对 DNA 结合域进行定点突变，R34M 突变蛋白完全丧失了对靶启动子的 DNA 结合活性。当 TRASH 结构域中 CXXC 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丧失了铜结合活性。因此，CopR 是一种铜感应转录调节因子，作为自身调节和嗜热球菌 NA1 中假定的铜外排系统 CopA 的抑制剂发挥作用。
