CopR binds and bends its target DNA: a footprinting and fluorescence resonance energy transfer study

Plasmid pIP501 encoded transcriptional repressor CopR is one of the two regulators of plasmid copy number. Previous data suggested that CopR is a HTH protein belonging to a family of 578 HTH proteins (termed HTH 3-family). Only a very limited number of proteins in this family, among them λ c1 repressor, 434 c1 repressor and P22 c2 repressor, have been characterized in detail so far. Previously, a CopR structural model was built based on structural homologies to the 434 c1 and P22 c2 repressor and used to identify amino acids involved in DNA binding and dimerization. Site-directed mutagenesis in combination with electrophoretic mobility shift assay (EMSA), dimerization studies and circular dichroism (CD) measurements verified the model predictions. In this study we used hydroxyl radical footprinting and fluorescence resonance energy transfer (FRET) measurements to obtain detailed information about the structure of the DNA in the CopR–DNA complex. Our results show that the DNA is bent gently around the protein, comparable to the bending angle of 20–25° observed in the 434 c1 repressor–DNA complex and the λ c1 repressor–DNA complex. The shape of CopR dimers as determined by sedimentation velocity experiments is extended and accounts for the relatively large area of protection observed with hydroxyl radical footprinting.