To demonstrate the regulatory activity of BreR, the authors cited the expression analysis done in their previous paper [PMID::18776020] (DNA expression array, B-gal assay) which showed BreR repressing both itself and breAB. From this starting point, they used the Vienna RNA Secondary Structure Prediction program to identify instances of dyadic symmetry in the promoter. They then used a combination of EMSAs and DNase I footprinting to screen these potential sites, and demonstrated that BreR has a dyadic binding site in its own promoter, as well as both distal and proximal dyadic binding sites in the promoter of breAB. The authors then used site-directed mutagenesis to confirm that these motifs were required for binding.
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|