EMSA confirmed CifR binding to the cifR-morB intergenic region. This binding was disrupted upon addition of the CifR substrate EBH. Additional EMSA experiment with two smaller, non-overlapping promoter fragments showed that cifR-morB intergenic region contained two CifR binding sites. By performing EMSA with smaller DNA fragments, the CifR binding sites were narrowed down further. morB and cifR transcription start sites were determined by 5'RACE. CifR binding was abolished after a 6 bp long mutation was inserted into the center of both putative sites. Western analysis showed that the same 6 bp long mutation in the morB proximal site caused an increase in Cif production. qRT-PCR performed using wild-type and 6 bp mutant strains confirmed that CifR represses morB and cifR gene expression.
Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.
|Site sequence||Regulated genes||Gene diagram||Experimental techniques||TF function||TF type|