Figure 5.

BrxR_Efer R17 is essential for transcriptional repression and DNA binding. (A) Close-up alignment of wHTH domains from the BrxR_Efer apo structure (cyan and salmon cartoons, PDB: 7QFZ), and BrxR_Acin-DNA structure (purple cartoon and DNA as sticks, PDB: 7T8K). Stabilization and recognition helices roughly overlay, and there is co-localization of BrxR_Efer R17 with BrxR_Acin R11. BrxR_Acin R11 makes bidentate hydrogen bonds with the DNA phosphate backbone. Distances shown are in angstroms. (B) LacZ-reporter assays using active pRW50-R7 construct with and without induction of His₆-BrxR_Efer or His₆-BrxR_Efer-R17A from pBAD30. Data are shown in triplicate, and error bars represent standard deviation of the mean. (C) Size exclusion chromatography of His₆-BrxR_Efer and His₆-BrxR_Efer-R17A resolved via a Superdex 200 increase GL 5/150 gel filtration column. Both proteins eluted at 1.84 ml, corresponding to a mass approximately twice their respective M_r, indicating correct folding into dimer formation. No additional peak was observed for residual monomers. (D) Electrophoretic mobility shift assay (EMSA) of titrated His₆-BrxR_Efer-R17A protein with WT dsDNA probe IR1-IR2 spanning pEFER nucleotide locations 12,801–12,870. Target probe was amplified to incorporate fluorescein and contains the native promoter region. Protein concentration is shown above each lane together with binding events (B – bound, U – unbound). Control lane of His₆-BrxR (WT) is included for comparison. Experiment was run in triplicate and a representative gel is shown.