Figure 3

Interactions between NnaR, GlnR or NnaR/GlnR mixture and putative promoter regions of msmeg_0427, msmeg_0433, msmeg_4008, msmeg_5360 and msmeg_5765 analysed by EMSA. For EMSA reactions (A), approximately 30 nM hexachlorofluorescein-labelled DNA was incubated with 0 or 2 µM NnaR, 4 µM GlnR or 2 µM NnaR combined with 4 µM GlnR for 10 min in EMSA reaction buffer containing 10 mM Tris, 100 mM KCl, 1 mM DTT, 2.5% glycerol, 20 mM MgCl₂, 500 ng poly(dI·dC), and 0.05% NP-40 (pH 7.5). (B) NnaR binding specificity to the promoter region of msmeg_0427 was analysed using the competition test. Approximately 30 nM hexachlorofluorescein-labelled DNA or a combination of both 30 nM labelled and 100-fold excess unlabelled probe was incubated with 2 µM NnaR for 10 min in EMSA reaction buffer. The samples were resolved in 2% agarose gel and visualized on a GE Typhoon 8600 Imager.