RisR Binding to Radiolabeled Promoters

Plasmids pPK14640 and pPK14641 were isolated using a QIAfilter maxi kit (Qiagen), and 30 ng was digested using BamHI and HindIII enzymes (NEB) to expose the 3′ ends of the fragment for radiolabeling with [α-³²P]dGTP (PerkinElmer) by Sequenase (Thermo Fisher). The relevant fragments were separated and isolated from a 5% acrylamide-TBE (Tris-borate-EDTA) gel using a QIAquick gel extraction kit (Qiagen). Promoter fragments were incubated with RisR (1 μM) for 25 min in 25 mM potassium phosphate buffer, 30 mM KCl, 5 mM potassium glutamate, 100 μg/mL BSA, and 1 mM DTT at 37°C under anaerobic conditions. DNase I (Worthington) 2 μg/mL in 65 mM MgCl₂ was added for 30 s, and the reaction was terminated with 300 mM acetate and 20 mM EDTA, ethanol precipitated and resuspended in 4 μL urea loading dye, heated to 90°C for 1 min before loading a 7-M urea −8.0% polyacrylamide gel in 0.5× TBE buffer. The G+A ladder for each radiolabeled promoter fragment was achieved by DNA modification using formic acid followed by piperidine cleavage (79 (link)). The gel was visualized in an Amersham Typhoon 5-gel imaging scanner (Cytiva). For some experiments, 5 μM RisR was treated with 32 units of enterokinase (NEB) to remove the N-terminal tag by 2 h of incubation at room temperature under anaerobic conditions in 50 mM potassium phosphate buffer, 100 mM NaCl, 2 mM CaCl₂, and 10% glycerol pH 7.2.