Studying Sigma–Core Interactions in Escherichia coli RNA Polymerase by Electrophoretic Shift Assays and Luminescence Resonance Energy Transfer

Abstract

Publisher Summary This chapter aims to describe electrophoretic mobility shift (EMS) assays and fluorescence resonance energy transfer (FRET) for several reasons. EMS assays are fairly simple and quick to perform with the equipment present in most biologically oriented laboratories. At the same time, they give useful initial information about a protein binding to another protein, DNA, or RNA. EMS assays are based on the change of mobility of a protein during polyacrylamide gel electrophoresis (PAGE) on binding to DNA, RNA, or another protein. Crucial to EMS assays the fact that the procedure involves separating the complex from the unbound binding partner by size and charge differences is discussed. This changes the equilibrium conditions at which initial binding occurs, and thus weak interactions, such as in complexes that have a half-life shorter than the time scale of the separation step, are under-represented, or cannot be detected, as the interaction does not persist throughout the procedure. It focuses on luminescence resonance energy transfer (LRET)-based assays for a homogeneous assay to measure formation of the σ70– β complex. LRET is a recent modification of FRET that uses a lanthanide-based donor fluorophore. The more general term luminescence instead of fluorescence indicates that lanthanide emission is technically not fluorescence.