Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae

Division of Biochemistry, Freie Universität Berlin, Berlín, Berlin, Germany
JBIC Journal of Biological Inorganic Chemistry (Impact Factor: 2.54). 07/2004; 9(4):396-402. DOI: 10.1007/s00775-004-0536-1
Source: PubMed


In Enterococcus hirae, copper homeostasis is controlled by the cop operon, which encodes the copper-responsive repressor CopY, the copper chaperone CopZ, and two copper ATPases, CopA and CopB. The four genes are under control of CopY, which is a homodimeric zinc protein, [Zn(II)CopY]2. It acts as a copper-responsive repressor: when media copper is raised, CopY is released from the DNA, allowing transcription to proceed. This involves the conversion of [Zn(II)CopY]2 to [Cu(I)2CopY]2, which is no longer able to bind to the promoter. Binding analysis of [Zn(II)CopY]2 to orthologous promoters and to control DNA by surface plasmon resonance analysis defined the consensus sequence TACAnnTGTA as the repressor binding element, or " cop box", of Gram-positive bacteria. Association and dissociation rates for the CopY-DNA interaction in the absence and presence of added copper were determined. The dissociation rate of [Zn(II)CopY]2 from the promoter was 7.3 x 10(-6) s(-1) and was increased to 5 x 10(-5) s(-1) in the presence of copper. This copper-induced change may be the underlying mechanism of copper induction. Induction of the cop operon was also assessed in vivo with a biosensor containing a lux reporter system under the control of the E. hirae cop promoter. Half-maximal induction of this biosensor was observed at 5 microM media copper, which delineates the ambient copper concentration to which the cop operon responds in vivo.

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    • "In this scenario, the decreased growth phenotype observed in bacteria lacking CopA could be due to reduced copper availability to the cuproproteins of the periplasm. The presence in E. hirae of a soluble copper-chaperone CopZ and a copper-dependent transcription factor CopY [12,13] may also indicate that CopA is a component of a regulatory network that provides copper to the regulator molecules in a biologically available form. "
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    • "This leads to the replacement of the Zn 21 cofactor of CopY by two Cu 1 ions and a concomitant decrease in DNA affinity, which in turn induces the expression of the cop operon (Strausak & Solioz, 1997; Cobine et al., 2002). The transfer of copper from CopZ to CopY involves protein–protein interaction , thereby conferring specificity to the process (Cobine et al., 1999; Portmann et al., 2004). CopZ was also shown to interact with the CopA copper ATPase, the presumed entry point of copper into E. hirae (Multhaup et al., 2001). "
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