In Vitro Transcription Assay

Transcription from promoter DNA fragments was performed essentially as described previously (12 (link), 19 (link)). Reactions were performed in 10 μl of transcription buffer TB (20 mM Tris HCl [pH 7.9], 40 mM KCl, and 10 mM MgCl₂) containing 1 pmol of coli RNAP core with 3 pmols of σ⁷⁰ and 10% DMSO with or without inhibitors. Transcription was initiated by the addition of a mixture of 25 μM CpA dinucleotide as a primer, 0.2 μl α-[³²P] UTP (10 mCi/ml; Hartmann Analytic), 10 μM UTP, 100 μM ATP, 100 μM CTP, and 100 μM GTP, and 10 nM promoter DNA. Reactions were stopped after 10-min incubation at 37°C by the addition of equal volume of formamide-containing loading buffer. Products were resolved in denaturing polyacrylamide gels, revealed by phosphorimaging (Cytiva), and analyzed using ImageQuant software (Cytiva).