Quantitative Dissection of RNA Polymerase-Promoter Interactions using Protein Beacon Assay

Abstract

Promoter recognition and melting by RNA polymerase (RNAP) are key points in gene expression and regulation. E. coli RNAP binding to promoter DNA and model promoter fragments can be measured using a new protein beacon assay. The assay relies on the detection of fluorescence signal from a fluorescent label incorporated into the σ70 subunit of RNAP close to region 2.3 of σ70, part of RNAP that recognizes the −10 promoter element. The ground level fluorescence of such RNAP beacon is low because the region 2.3 aromatic amino acids quench the fluorescence. When RNAP beacon binds promoter, the quenching interactions become destroyed, leading to increased fluorescence.Promoter melting in bacteria is nucleated at upstream edge of the transcription bubble. The mechanism of downstream propagation of the transcription bubble to include the transcription start site is unclear. We introduced new downstream fork junction promoter fragments mimicking the downstream segment of promoter complexes. We demonstrated that RNAP binding to downstream fork junctions was coupled with DNA melting around the transcription start point and identified structural determinants of affinity and transcription activity of RNAP-downstream fork junction complexes.The product of E. coli T7 bacteriophage gene 2 (gp2 protein) is a potent inhibitor of host RNAP. We applied the beacon assay to the mechanism of gp2 inhibition. We measured the effect of gp2 on RNAP binding to various promoter fragments. In this way, the effect of gp2 on RNAP-promoter interactions was dissected. Gp2 greatly decreased RNAP affinity to downstream promoter duplex and inhibited RNAP binding to template and non-template strand segments located between the −10 promoter element and downstream edge of the transcription bubble. The inhibition of RNAP interactions with the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism.