IscR-C92A Protein Binding Assay

E. coli IscR-C92A protein that lacks the [2Fe-2S] cluster was isolated as previously described [25 (link), 30 (link)] and subsequently used in electrophoretic mobility shift assays (EMSAs) because this apo- form of IscR binds exclusively to type II sites. DNA fragments containing the wild-type Y. pseudotuberculosis lcrF promoter (-206 to +12 bp relative to the +1 transcription start site), or its lcrF^pNull variant in which the IscR binding site is disrupted, were isolated from pPK12778 and pPK12779, respectively, after digestion with HindIII and BamHI, and EMSAs were carried out with purified IscR-C92A as previously described [29 (link)]. Cut vector backbone is also present in the reaction and serves as a source of nonspecific DNA. After incubation at 37°C for 30 min, samples were loaded onto a non-denaturing 6% polyacrylamide gel in 0.5× Tris-borate-EDTA buffer and run at 100 V for 4.0 hrs at room temperature. The gel was stained with SYBR Green EMSA nucleic acid gel stain (Molecular Probes) and visualized using a Typhoon FLA 900 imager (GE).