Interaction of a repressor and its binding sites for regulation of the Bacillus subtilis iol divergon

Transcription of the Bacillus subtilis iol divergon is negatively regulated by a repressor encoded by iolR, which belongs to the DeoR family of bacterial regulators. Gel retardation analysis involving the IolR protein synthesized in Escherichia coli revealed that IolR bound specifically and independently to each of the iol and iolRS promoter regions, with higher affinity to iol. DNase I footprinting revealed that IolR affected DNase I sensitivity either in the iol promoter region between nucleotides -46 and +51 or in iolRS between -79 and -2 (+1 is the transcription initiation nucleotide of both iol and iolRS), indicating its interaction with the extended regions of the iol and iolRS promoters. Deletion analysis indicated that the iol region between -23 and +21 is involved mainly in IolR binding and negative regulation, while the iolRS region between -70 and -44 comprises at least part of the cis-acting sequences for IolR binding and negative regulation. Sequence examination of the extended regions revealed that a tandem direct repeat consisting of two relatively conserved 11-mer sequences, WRAYCAADARD (where D is A, G or T; R is A or G; W is A or T; and Y is C or T), found in each of the iol and iolRS regions might be a determinant sequence for the IolR-DNA interaction. Actual involvement of the direct repeats in the IolR-DNA interaction was shown by the deficiency of IolR-binding and negative regulation that was caused by substitution of the conserved bases within the conserved sequences. These results imply a unique mode of interaction of IolR with the target DNA.