Abstract
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.
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Acknowledgements
We thank Drs Kanai T and Atomi H for in vitro transcription assay with transcription factors of Thermococcus kodakarensis at Kyoto University. This work was supported by the ‘Biohydrogen production from hyperthermophiles’ program of the Korea Institute of Ocean Science and Technology (KIOST) and by the National Research Foundation of Korea (2010-0010779).
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Kim, SY., Jeong, H.J., Kim, M. et al. Characterization of the copper-sensing transcriptional regulator CopR from the hyperthermophilic archeaon Thermococcus onnurineus NA1. Biometals 32, 923–937 (2019). https://doi.org/10.1007/s10534-019-00223-2
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DOI: https://doi.org/10.1007/s10534-019-00223-2