A synthetic biology approach to bacterial transcription initiation: RNA aptamer based in vitro transcription assay for rapidly testing bacterial RNA polymerases, promoters and inhibitors.

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 Escherichia coli RNA polymerase; notably, E. coli enzyme activity remained strictly dependent on the presence of a {sigma}-factor and a cognate promoter sequence. To maximize signal-to-noise ratios, we incorporated two rrnBT1 terminators upstream of the promoter of interest. This modification successfully eliminated background transcription in case of weak promoters (PlivJ) and prevented interference from read-through transcription in strong synthetic promoters (Ptrc*). 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.