In Vitro Transcription Assay

Transcription from promoter DNA fragments was performed essentially as described (20 (link)). Briefly, reactions were performed in 10 μl of transcription buffer TB (20 mM Tris–HCl pH 7.9, 40 mM KCl, 10 mM MgCl₂). 1 pmol of E. coli RNAP core with 3 pmol of σ⁷⁰ or 1 pmol of T. aquaticus RNAP core with 3 pmol of T. aquaticus σ^A or 1 pmol of S. aureus RNAP core with 3 pmol of S. aureus σ^A or 1 pmol of M. smegmatis or S. epidermidis RNAP holoenzymes were incubated in TB with 1 μl of DMSO containing or not containing Urd at 37°C (or 60°C in case of T. aquaticus RNAP) for 5 min. Transcription was initiated by the addition of 2 μl mixture of nucleotides and promoter DNA in TB, containing (final concentrations): 10 nM promoter DNA, 25 μM CpA (for T7A1 and GalP1 promoters) or 100 μM ApA (for lacUV promoter), 0.2 μl α-[³²P]UTP (10mCi/ml) (Hartmann Analytic), 10 μM UTP with (run off transcription) or without (abortive transcription) 100 μM ATP, CTP and GTP. Reactions were stopped after 10 min incubation at 37°C (or 60°C in case of T. aquaticus RNAP) for run off transcription or 5 min for abortive transcription by the addition of equal volume of formamide-containing loading buffer. Products were resolved in denaturing polyacrylamide gels, revealed by PhosphorImaging (GE Healthcare), and analyzed using ImageQuant software (GE Healthcare)