A Multiwell Assay to Isolate Compounds Inhibiting the Assembly of the Prokaryotic RNA Polymerase

Abstract

We have developed a multiwell assay for the detection of modulators of prokaryotic transcription based on the quantification of protein–protein interaction. This assay consists of three steps: (a) the immobilization of the Escherichia coli protein σ⁷⁰ in the well, (b) the incubation of the immobilized protein with core RNA polymerase and a potential inhibitor, and (c) washing and quantification of the binding of core to σ⁷⁰ with a monoclonal antibody conjugated to horseradish peroxidase. We show that this assay is sensitive, reproducible, and robust, and is able to discriminate between control competitors with different affinities. We demonstrate the usefulness of the assay to screen for microbial RNA polymerase inhibitors as potential new drugs for the treatment of emerging antibiotic-resistant bacteria.