Figure 4.

CodY interaction with the regulatory region of tpxD. EMSA assay was used to confirm the presence of CodY binding site in tpxD regulatory region: Effect of H₂O₂ (A) and BCAA (B). 10 nM of fluorescently labeled DNA probe was mixed with increasing concentration of recombinant CodY, and incubated for 30 min at room temperature. When required, 2 mM H₂O₂ and/or BCAA (isoleucine 1.62 mM, leucine 3.48 mM, and valine 2.77 mM) were added into the binding buffer. DNA-protein complexes were separated by electrophoresis on non-denaturing PAGE (8%). In all gels, Lane 1 contains DNA probe alone, indicated with a black arrow. DNA-protein interaction is seen as an upward shift of DNA probe, indicated with a dotted arrow. (C) Lane 1, 50 ng approximately of the 81 bp DNA fragment alone, lane 2 and 3, DNA plus 0.6- and 1-mM recombinant CodY, respectively. Lane 4, DNA probe, excluding 15 bp putative CodY binding site plus 1 mM recombinant CodY. White arrow indicates the position of DNA-protein complex. EMSA was performed using Molecular Probes fluorescence-based EMSA kit (Invitrogen). Briefly, 5X FY binding buffer (20 mM Tris-HCl pH 7.5, 30 mM KCl, 1 mM DTT, 1 mM EDTA pH 8.0 and 10% v/v glycerol) was prepared to incubate the promoter probe and protein. The binding reaction was set up by mixing a constant amount of target promoter probe (~30 ng), and increasing amounts (0.1–0.5 μM) of purified and dialyzed His-tagged protein. The binding reaction was incubated at room temperature for 20 min in a total volume of 20 μl, and then analyzed on an 8% w/v non-denaturing polyacrylamide gel. After electrophoresis, gels were stained with SYBR® Green EMSA gel stain (Invitrogen) and visualized using a Typhoon Trio+ scanner (GE Healthcare Life Sciences) with a 526 nm short-pass wavelength filter.