CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases

Abstract

The cop operon of Enterococcus hirae effects copper homeostasis in this organism. It encodes a repressor, CopY, an activator, CopZ, and two P-type copper ATPases, CopA and CopB. Expression of all four genes is regulated by the ambient copper. In this regulation, CopY apparently acts as a copper- inducible repressor. By DNase I footprinting, it was shown that purified CopY protected two discrete sites in the region encompassing nucleotides -71 to - 11 relative to the translational start site and containing hyphenated inverted repeats. Transcription is initiated between these repeats at nucleotide -42, in a domain that remained accessible to DNase I in the DNA- repressor complex. Chemical cross-linking revealed that CopY exists as a dimer in solution. In DNA band-shift assays, it was apparent that the CopY- DNA interaction occurred in two discrete steps. Half-maximal binding of repressor to the two operator sites was observed at 2 x 10-9 M and 5 x 10-9 M CopY, respectively. Copper ions released CopY from the promoter/operator with an apparent half-binding constant for Cu(I) of 20 μM. The site-directed mutations A-61T and A-30T essentially abolished the binding of CopY to the respective binding sites, and the double mutation A-61T/A-30T inactivated both binding sites. Thus, CopY is a copper-inducible repressor of the cop operon of E. hirae, exhibiting highly specific DNA-protein interactions with two sites on the cop promoter/operator and playing a key role in copper homeostasis in E. hirae.