Studying the interaction of the Escherichia coli sigma factors with core RNA polymerase using LRET

The process of transcription in Escherichia coli is a multistep process that results in the production of RNA from a DNA-template. The transcription machinery is composed of a DNA-dependent core RNA polymerase (RNAP) that can produce RNA transcripts but cannot recognize specific promoters. When core RNAP binds to one of seven sigma factors to form the holoenzyme, it can then be directed to a distinct set of promoters in response to different growth conditions and stresses. Understanding the interaction of these sigma factors with core RNAP can provide insights into how global transcription is regulated.;In this work we used the LRET assay, a homogenous fluorescence-based binding assay, to study the interactions of sigma70 and sigma32 with core RNAP. The differential responses of these interactions in NaCl and Kglu were measured. In all conditions tested sigma32 bound core RNAP more strongly (KD ∼ 0.5-2 nM) than sigma70 (KD ∼ 3-300 nM), however, the interaction of these sigma factors with the beta'coiled-coil (beta'cc), a major site of sigma interaction with core RNAP, was found to be stronger for sigma70 compared to sigma32. Dissociation studies revealed that sigma32 had a half-life of dissociation that was much slower than sigma70 (about 10-fold). Both sigma70 and sigma32 were found to form increasing levels of a non-dissociable form of holoenzyme upon incubation. These data suggest that the competition for the binding of sigma factors to core may be regulated by association rates and protein concentration rather than through a rapid equilibrium for binding.;The interaction of sigma70 with core RNAP is an attractive target for the discovery of new antibiotics. A high throughput screen for small molecule inhibitors of the interaction of sigma70 with the beta'cc was performed. Four hits from a library of 16,000 compounds were found that had high potency and inhibited in vitro transcription. Characterization and structure-activity relationship experiments were performed to determine the usefulness of these compounds as possible lead candidates for antibiotic discovery.