Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae.
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.
Portmann
Reto
R
Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, 3010 Berne, Switzerland.
Magnani
David
D
Stoyanov
Jivko V
JV
Schmechel
Ariane
A
Multhaup
Gerd
G
Solioz
Marc
M
D001426:Bacterial Proteins / Q000378:metabolism* / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods; D017124:Conserved Sequence; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology; D016983:Enterococcus / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism* / Q000502:physiology
D001426:Bacterial Proteins; C092378:CopY protein, Enterococcus hirae; D012097:Repressor Proteins; D003300:Copper