CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases.
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 microM. 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.
Strausak
D
D
Department of Clinical Pharmacology, University of Berne, 3010 Berne, Switzerland.
Solioz
M
M
D000251:Adenosine Triphosphatases / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D027682:Cation Transport Proteins; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D016983:Enterococcus; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; C092378:CopY protein, Enterococcus hirae; D004269:DNA, Bacterial; D012097:Repressor Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases