Protein-protein interactions between CbbR and RegA (PrrA), transcriptional regulators of the cbb operons of Rhodobacter sphaeroides

CbbR and RegA (PrrA) are transcriptional regulators of the cbb(I) and cbb(II) (Calvin-Benson-Bassham CO2 fixation pathway) operons of Rhodobacter sphaeroides. Both proteins interact specifically with promoter sequences of the cbb operons. RegA has four DNA binding sites within the cbbI promoter region, with the CbbR binding site and RegA binding site 1 overlapping each other. This study demonstrated that CbbR and RegA interact and form a discrete complex in vitro, as illustrated by gel mobility shift experiments, direct isolation of the proteins from DNA complexes, and chemical cross-linking analyses. For CbbR/RegA interactions to occur, CbbR must be bound to the DNA, with the ability of CbbR to bind the cbbI promoter enhanced by RegA. Conversely, interactions with CbbR did not require RegA to bind the cbbI promoter. RegA itself formed incrementally larger multimeric complexes with DNA as the concentration of RegA increased. The presence of RegA binding sites 1, 2 and 3 promoted RegA/DNA binding at significantly lower concentrations of RegA than when RegA binding site 3 was not present in the cbbI promoter. These studies support the premise that both CbbR and RegA are necessary for optimal transcription of the cbbI operon genes of R. sphaeroides.