Abstract
<jats:p> We present a robust and versatile in vitro transcription (IVT) assay utilizing an optimized Broccoli RNA aptamer sequence. When paired with the fluorophore DFHBI-1T, this system enables real-time monitoring of multi-round transcription over several hours. We developed a dedicated plasmid backbone, pIVT3, designed for the streamlined insertion of diverse promoters. The system was validated using single-subunit T7 RNA polymerase and multi-subunit <jats:italic>Escherichia coli</jats:italic> RNA polymerase; notably, <jats:italic>E. coli</jats:italic> enzyme activity remained strictly dependent on the presence of a σ-factor and a cognate promoter sequence. To maximize signal-to-noise ratios, we incorporated two <jats:italic>rrnB</jats:italic> T1 terminators upstream of the promoter of interest. This modification successfully eliminated background transcription in case of weak promoters (P <jats:italic>livJ</jats:italic> ) and prevented interference from read-through transcription in strong synthetic promoters (P <jats:italic>trc*</jats:italic> ). Furthermore, we demonstrated the assays utility in drug discovery by testing the RNA polymerase inhibitor rifampicin, which showed time- and dosage-dependent inhibitory kinetics. Collectively, our results establish this Broccoli-based IVT system as a highly adaptable platform for quantifying promoter strength and screening small-molecule inhibitors of bacterial transcription. </jats:p>