KMnO4 Footprinting.

A linear DNA fragment containing the λP_R promoter was made by PCR amplification on pIA226 with primers 17 and 256 (Table S2); the NT strand primer (no. 17) was end-labeled with [γ³²P]-ATP using polynucleotide kinase (PNK) (New England Biolabs) and purified using G-50 spin columns (GE Healthcare). Sequencing reactions were performed with [³²P]-labeled primer no. 17 with the Sequenase version 2.0 DNA sequencing kit (Affymetrix USB). Open complexes were formed with RNAP (40 nM) and the labeled DNA template (30 nM) for 15 min at 37 °C in GBB buffer (20 mM Tris⋅HCl, 20 mM NaCl, 14 mM MgCl₂, 5% glycerol, and 0.1 mM EDTA; pH 7.9). Samples were shifted to room temperature and treated with 2 mM KMnO₄ for 30–60 s. Aliquots were quenched by the addition of 2.5 μL of β-mercaptoethanol and 2.5 μL of 0.5 M EDTA. The volume was adjusted to 100 μL with H₂O. Samples were subjected to phenol-chloroform extraction. Fifty microliters of GEA mix (1 mg/mL glycogen, 25 mM EDTA, 0.3 M Na acetate) were added, followed by 2.5 volumes of ethanol. Pellets were dissolved in 20 µL H₂O and incubated with 100 µL of 0.5 M piperidine at 95 °C for 12 min. After the addition of 70 μL GEA and ethanol precipitation, DNA was dissolved in 96% formamide containing 0.1% xylene cyanol and 0.1% bromophenol blue.