Electrophoretic Mobility Shift Assay (EMSA)

Electrophoretic mobility shift assays (EMSAs) were performed using the LightShift^TM optimization and control kit (Thermo Scientific) according to the manufacturer’s protocol and as previously described (Batte et al., 2016; Pandey et al., 2019). Briefly, the binding reaction mixture was prepared with ultrapure water containing 1× binding buffer, 50 ng poly (dI-dC), 2.5% (v/v) glycerol, 0.05% (v/v) NP-40, and 5 mM MgCl₂. Biotin-labeled single-stranded DNA (ssDNA) upstream of the transcription initiation site of the ndh₂ gene and double-stranded DNA (dsDNA) of the ccpE gene in the appropriate concentrations were incubated with increasing concentrations of purified recombinant MsaB–His protein in the binding reaction buffer and unlabeled specific probe when required. The mixture was then incubated at room temperature for 25 min and subjected to electrophoresis at 100 V for 1 h in a pre-run 5% Tris-borate EDTA (TBE) gel. The samples were then transferred to a nylon membrane, incubated for 45 min in the cold, crosslinked, and processed for the detection of samples. The protein–DNA complexes in the gel were then visualized using the Chemiluminescent EMSA kit (Thermo Scientific) according to the manufacturer’s protocol and imaged with a ChemiDoc system (Bio-Rad). The probes used for EMSA studies are listed in Supplementary Table 2.