Abortive Transcription Gel for WhiB7 paper
Abortive transcription assays of Mycobacterium tuberculosis RNAP holoenzyme with transcription factors RbpA and CarD on the whiB7 promoter. Samples is a triplication experiment with and without the transcription factor WhiB7. Data analyzed with ImageJ.
Steps to reproduce
Transcription assays were performed with 50 nM of Mtb RNAP holoenzymes with or without WhiB7 in transcription buffer [10 mM Tris HCl, pH 7.9, 80 mM K-acetate, 10 mM, MgCl2, 1 mM DTT, 5 μg/ml bovine serum albumin (BSA) and 0.1 mM EDTA]. Both holoenzymes were prepared as described in protein preparation for cryo-EM grids. After purification on Superose 6 Increase 10/300 GL column (GE Healthcare, Pittsburgh, PA), holoenzymes were mixed with 250 nM CarD while the RNAP/WhiB7 holoenzyme was mixed with 250 nM RbpA. The whiB7 promoter (-70 to +30) was then added (10 nM) to the holoenzymes, and the samples were incubated for 15 min at 37 °C to allow the formation of RNAP open complex. Transcription was initiated by adding a nucleotide mixture consisting of 250 μM CpG dinucleotide (Trilink Biotechnologies, San Diego, CA), 50 μM CTP, and 1.25 μCi (15 nM)- [-P32 ] CTP. Each reaction was allowed to proceed for 10 min at 37 °C, and reactions were quenched by the addition of 2x stop buffer (0.5X TBE, pH 8.3, 8 M urea, 30 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol). Reactions were heated to 95°C for 10 min and loaded onto a polyacrylamide gel [23% Acrylamide/Bis acrylamide (19:1), 6M urea, and 1X TBE, pH8.3]. Transcription products were visualized by using Typhoon 9400 Variable Imager (Amersham Biosciences) and quantified using Image J(Schneider et al., 2012). Quantified values were plotted in a histogram and the mean standard errors were calculated from three independent data sets.