Plasmid pIP501 encoded transcriptional repressor CopR binds asymmetrically at two consecutive major grooves of the DNA.

Replication of the streptococcal plasmid pIP501 is regulated by the CopR protein and an antisense-RNA (RNAIII). CopR acts as transcriptional repressor at the essential repR promoter pII by binding to inverted repeat IR1 upstream of pII. To further characterize the interaction of CopR with its target, footprinting studies were performed. Methylation interference identified three guanine bases (G240, G242 and G251) in the top strand and two (G252 and G254) in the bottom strand contacted by CopR in the major groove of the DNA. Missing base interference revealed the contribution of the bases in the neighbourhood of these guanine bases to the specific DNA-protein contacts. Phosphate residues essential for CopR binding were determined by ethylation interference. The recognition sequence was localized at the centre of inverted repeat IR1. CopR contacts two consecutive major grooves (site I and II) on the same face of the DNA. Although the two sites share a common sequence motif, neighbouring bases are contacted differently. DNA fragments carrying single mutations in site I or II were analysed by band shift assays. Gel filtration and native gel electrophoresis demonstrated that CopR exists only as a dimer. A sigmoidal binding curve of CopR to its DNA target was observed and allowed the determination of the apparent dissociation constant K(D). The significance of the relatively high apparent K(D) for the role of CopR in pIP501 copy number regulation is discussed.