Fig. 5
In vitro electrophoretic mobility-shift assay (EMSA) assay of RimR2 binding to the promoter regions of the rimocidin biosynthetic genes rimA(a), rimC (b), rimD (c), rimF (d), rimG (e), rimH (f), rimR1 (g), and its own gene rimR2 (h). The 5′-biotin labeled DNA probe containing tested promoter regions were incubated with His6-tagged RimR2 protein. A 100-fold excess of unlabeled specific competitor was added to the competition assay, respectively. RimR2 protein binding putative promoter region of rimA gene (a), rimC gene (b), rimD gene (c), rimF gene (d), rimG gene (d), rimH gene (d), rimR1 gene (g), rimR2 gene (h). The symbols “ + ” or “ − ”in the top row indicate the presence or absence of probes and competitors. Lane 1: biotin-labeled DNA probe; lane 2: biotin-labeled DNA probe plus RimR2 protein; lane 3: a 100-fold excess of unlabeled specific competitor plus RimR2 protein. All binding experiments were performed using 0.04 pmol/μl of biotin-labeled DNA probe and 10 μg of RimR2 protein