Gelcode

For photo-crosslinking using σD581BpA and wt core in Figure 3, 38 pmol of σ (in 1 μl TGED/NaCl/Triton) were incubated with 1 μl AsiA buffer [10 mM Tris-Cl (pH 8), 0.1 mM EDTA, 50% glycerol, 500 mM NaCl, 0.1 mM DTT] with or without 65 pmol AsiA and 1 μl of 5× Kglu transcription buffer lacking BSA [40 mM Tris-acetate (pH 7.9), 150 mM potassium glutamate, 4 mM magnesium acetate, 0.1 mM EDTA, and 0.1 mM DTT] for 10 min at 37°C in 1.5 ml eppendorf tubes. RNAP core (4 pmol in 2 μl RNAP storage buffer) or buffer alone was then added and the solution incubated for 10 min at 37°C before collection on ice. Tubes were laid flat on the UV lamp for a total of 30 min at room temperature, turning the tubes to the opposite side after 15 min. Samples were electrophoresed on 10–20% Tris-tricine gels (Invitrogen) and stained with Colloidial Coomassie Blue (Invitrogen). Photocrosslinking reactions in Figure 6B were assembled similarly except reactions contained 120 pmol σD581BpA, 400 pmol AsiA, and 15 pmol of wt or mutant core in a total volume of 30 μl. A 20 μl aliquot was used for photocrosslinking, and a 5 μl aliquot was applied to a 4–12% Tris-glycine gel (Invitrogen/Thermo Fisher) run in 1× Native Tris-glycine buffer (Invitrogen/Thermo Fisher) and stained in Gel Code (Thermo Fisher) as described (46 (link)).