Figure 4: Inhibitory effects of drugs on the DNA-affinity of RamR.

(a) EMSA for RamR binding to DNA. The labelled DNA (15.5 fmol) was incubated with increasing concentrations (0, 15, 20, 25 or 30 pmol) of the purified RamR protein (upper left). To test the inhibitory effect of berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G on DNA–RamR binding, each drug was added to an incubation mixture containing the labelled DNA and 20 pmol RamR, at the concentrations indicated. Tetracycline was used as a negative control. (b) Binding of RamR to DNA analysed by SPR. DNA was immobilized onto a sensor chip, and then purified RamR protein was injected at different concentrations as indicated. (c) Inhibitory effects of drugs on RamR and DNA binding calculated from the results of SPR analysis. DNA was immobilized onto a sensor chip, and then the purified RamR protein (2 μM) was run over the sensor surface in the presence or absence of drugs at the various concentrations indicated. Percentage bound was calculated from the amount of RamR binding with DNA in the absence of drug, which was assigned as 100%. Shown is the result of one of the three experiments, which gave similar results.