Abortive Transcription Gel for Sven paper

Published: 30 August 2022| Version 1 | DOI: 10.17632/84gck7pypg.1
Contributor:
Mirjana Lilic

Description

Abortive transcription assays of Streptomyces venezuelae RNAP holoenzyme with transcription factors WhiA, WhiB, and RbpA on the sepX promoter. Samples is a triplication experiment with holoenzyme without transcription factors WhiA and WhiB, holoenzyme with only WhiA, holoenzyme with only WhiB, or holoenzyme with both transcription factors, WhiA and WhiB. Data analyzed with ImageJ.

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Transcription assays were performed with 50 nM of Sven RNAP holoenzymes in transcription buffer [10 mM Tris HCl, pH 7.9, 50 mM K-glutamate, 10 mM, MgCl2, 1 mM DTT, 5 μg/ml bovine serum albumin (BSA) and 0.1 mM EDTA]. All holoenzymes were prepared as described in the section with the assembly of holoenzymes. After purification on Superose 6 Increase 10/300 GL column (GE Healthcare, Pittsburgh, PA), holoenzymes with or without WhiB protein were mixed with 200 nM WhiA when WhiA was present in the reaction. Four holoenzyme samples (without transcription factors, only with WhiA, only with WhiB, and with both factors) were mixed with 10 nM sepX promoter (-70 to +30) 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 GpU dinucleotide (Trilink Biotechnologies, San Diego, CA), 50 μM GTP, and 1.25 μCi (15 nM)- [-P32 ] GTP. 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.

Institutions

Rockefeller University

Categories

Natural Sciences

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