Purification and characterization of the CopB replication control protein, and precise mapping of its target site in...

The CopB regulatory loop from plasmid R1 has been analyzed. The CopB protein was partially purified, but proteolytic activity in vitro resulted in the recovery of two molecular forms of the polypeptide. Both of these acted as repressors of the repA promoter and had identical activities. The smaller of the proteins was found to be the result of a specific cleavage in the normal in vivo translation product. The active form of the CopB protein is most likely a tetramer, which binds to a DNA region overlapping the repA promoter that also contains a stretch of dyad symmetry. Footprinting analysis and mutant analysis (including nucleotide sequence determination) identified this binding site within 20-25 base pairs. In agreement with in vivo results the binding between CopB and its target site is moderate compared with other operons like lac and trp.