In Vitro RNAP Transcription Assay

Run-off in vitro transcription was performed as previously described (54 (link)). Briefly, purified RNAP was incubated with different purified σ factors in a 1:5 molar ratio in transcription buffer (10 mM Tris–HCl pH 8.0, 10 mM MgCl₂, 0.5 mM dithiothreitol (DTT), 50 mM KCl, 500 μg/ml acetylated bovine serum albumin, 5% glycerol) and incubated on ice for 15 min. The in vitro transcription reactions contained 10 nM of linear promoter fragment in transcription buffer and 40 nM of RNAP. After 10 min of incubation 37°C, transcription was initiated by adding 0.25 mM (final concentration) of adenosine triphosphate (ATP), guanosine triphosphate (GTP) and cytidine triphosphate (CTP) and 0.025 mM uridine triphosphate (UTP) and 25 μCi of [α-³²P]-UTP. After 10 min of incubation, the reaction products were ethanol precipitated in the presence of 2 mM ethylenediaminetetraacetic acid, 0.3 M sodium acetate pH 5.2 and 3 μg glycogen. The RNA pellet was washed with 70% cold ethanol, dried and dissolved in formamide-containing loading buffer and separated on a 6% denaturing polyacrylamide sequencing gel. The gel was then dried and exposed to a phosphorimager screen. The resulting phosphorimage was visualized using a Typhoon FLA 7000 imaging system (GE Healthcare) and analyzed using ImageJ software.