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 sigma70 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 sigma70 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.
Our results demonstrate that this new molecule is active; its effects are fast and kinetically related to those of rifampicin, but unlike rifampicin it does not select for resistant bacteria.
We have recently isolated a monoclonal antibody directed against Escherichia coli RNA polymerase that does not inhibit transcription. This antibody is a useful tool to immobilize this enzyme for transcription assays or protein-protein interaction studies. The epitope of this monoclonal antibody was precisely located by a combination of protein deletion and synthetic peptide scanning. The amino acids of the epitope were also determined. We conclude that this antibody binds an epitope shared by several bacterial species and therefore can be used to characterize or purify other related enzymes.
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