Use of RNA polymerase molecular beacon assay to measure RNA polymerase interactions with model promoter fragments

Abstract

RNA polymerase–promoter interactions that keep the transcription initiation complex together are complex and multipartite, and formation of the RNA polymerase–promoter complex proceeds through multiple intermediates. Short promoter fragments can be used as a tool to dissect RNA polymerase–promoter interactions and to pinpoint elements responsible for specific properties of the entire promoter complex. A recently developed fluorometric molecular beacon assay allows one to monitor the enzyme interactions with various DNA probes and quantitatively characterize partial RNA polymerase–promoter interactions. Here, we present detailed protocols for the preparation of an Escherichia coli molecular beacon and its application to study RNA polymerase interactions with model promoter fragments.