BINDING CHARACTERISTICS OF ESCHERICHIA-COLI BIOTIN REPRESSOR OPERATOR COMPLEX

The genes of the operon bioA.BFCD are transcribed divergently from a single regulatory region between the bioA and, bioB genes. Transcription in both directions is co-repressed by biotinyl-5'-adenylate and the biotin repressor. The multimeric state of the biotin repressor bound to DNA and how it affects transcription have not been fully characterized. Therefore, we isolated the BirA protein from a recombinant strain which overproduced the biotin repressor and studied the repressor-operator binding characteristics through restriction enzyme site protection experiments and the mobility shift assay. We also measured the stoichiometry of the biotin repressor-operator complex directly. The results of restriction enzyme site protection studies are consistent with the postulation that the biotin operator is approx. 40 bp long. Only one retarded DNA band appeared in the mobility shift experiments, suggesting that the repressor-operator binding could be a single step reaction. The repressor-operator binding stoichiometry determination revealed that two repressor monomers may occupy the wild type or half palindromic biotin operator sequences.