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Abstract
The bacterial RNA polymerase (RNAP) is a large, complex molecular machine that is
the engine of gene expression. Despite global conservation in their structures and
function, RNAPs from different bacteria can have unique features in promoter and transcription
factor recognition. Therefore, availability of purified RNAP from different bacteria
is key to understanding these species-specific aspects and will be valuable for antibiotic
drug discovery. Pseudomonas aeruginosa is one of the leading causes of hospital
and community acquired infections worldwide - making the organism an important public
health pathogen. We developed a method for producing high quantities of highly pure
and active recombinant P. aeruginosa str. PAO1 RNAP core and holoenzyme complexes
that employed two-vector systems for expressing the core enzyme (α, β, β’, and ω subunits)
and for expressing the holoenzyme complex (core + σ 70 ). Unlike other RNAP expression
approaches, we used a low temperature autoinduction system in E. coli with T7 promoters
that produced high cell yields and stable protein expression. The purification strategy
comprised of four chromatographic separation steps (metal chelate, heparin, and ion-exchange)
with yields of up to 11 mg per 500 mL culture. Purified holoenzyme and reconstituted
holoenzyme from core and σ 70 were highly active at transcribing both small and large-sized
DNA templates, with a determined elongation rate of ~18 nt/s for the holoenzyme. The
successful purification of the P. aeruginosa RNAP provides a gateway for studies
focusing on in vitro transcriptional regulation in this pathogen.