Conversion of 3,3′,5,5′-tetramethylbenzidine/H2O2 substrate detected the presence of rDnrO. A Bio-Rad microplate reader recorded colorimetric readings at

450 nm. The inhibitory effect of DNR on the DNA–DnrO interaction was shown by EMSA in a nondenaturing PAGE. Purified rDnrO protein retarded the mobility of 150-bp DNA that has the 37-bp sequence in the middle (Lanes 2–4 in Fig. 1). However, there was no mobility shift in the presence of 2 ng DNR. This suggested that DNA–DnrO complex formation was hindered by intercalation of DNR to DNA (Lanes 5–7 in Fig. 1). The DNA–DnrO complex formation is essential for activation of dnrN (Otten et al., 2000). Increase in intracellular DNR level therefore determines whether DnrO can bind to its cognate sequence. An earlier study speculated that inhibition of DNA–DnrO interaction could be due to the formation of inhibitory complex with DNR (Jiang & Hutchinson, 2006). Inhibition Dinaciclib chemical structure of JadR and RedZ autoregulation has been shown in S. coelicolor, in which jadomycin and undecylprodigiosin bind to these transcription factors to inhibit transcription (Wang et al., 2009). These data prompted us to investigate the possible interaction of DnrO and DNR using an ultrafiltration technique. The pigmented DNR was mixed with rDnrO at pH 7.2 and at a temperature of 37 °C. The mixture was passed through a 10-kDa cut-off membrane, which retained the 38-kDa protein and passed the drug. There

was no fluorescence emission (590 nm) for DNR in the retentate (data not shown). The experiment was performed

alongside a known DNR-binding Obeticholic Acid Sitaxentan protein that served as positive control (Prasad et al., 2003). Therefore it was concluded that DnrO does not interact with DNR, and that the DNA binding by DnrO is inhibited due to DNR intercalating to DNA. The 37-bp DnrO-binding sequence that has GC-rich stretches was probed for the presence of DNR-intercalating sites. It has been theoretically estimated that on average, a molecule of DNR intercalates once in every 300 bp in calf thymus DNA (Chen et al., 1986) and prefers GC-rich DNA (Moore et al., 1989; Cullinane et al., 1994). DNA–DNR interaction has been extensively studied using various biophysical methods (Manfait et al., 1982) and its role as an inhibitor for transcription has been established (Straney & Crothers, 1987). DNR intercalation is an important element for this organism, as it produces the drug and yet survives its antibiotic properties. In silico analysis identified three high-affinity DNR intercalation sites in the 37-bp DNA. As shown previously, all these were sequences containing GG, GC and GA. The energy values were −13.6, −12.7 and −12.4 kcal mol−1, respectively (Fig. 2). The negative energy values indicate spontaneous intercalation of DNR with DNA. Similar DNR-intercalating motifs have been reported in dnrI promoter, which inhibits DnrN binding in the presence of DNR (Furuya & Hutchinson, 1996), but the mechanism has not yet been studied.