Abortive Transcription Gel for WhiB7 paper
Description
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.
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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.