Fluorescence Resonance Energy Transfer (FRET) in Analysis of Transcription-Complex Structure and Function

Abstract

Publisher Summary This chapter discusses the Fluorescence Resonance Energy Transfer (FRET) to monitor movement of RNA polymerase (RNAP) relative to DNA during transcription and to define three-dimensional structures of transcription complexes in solution. FRET is a physical phenomenon that permits measurement of distances. FRET occurs in a system where a fluorescent probe serves as a donor and a second fluorescent probe serves as an acceptor, where the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. The chapter presents protocols for FRET experiments that permit measurement of distances between positions on upstream DNA and positions within RNAP (“trailing-edge FRET”), distances between positions on downstream DNA and positions within RNAP (“leading-edge FRET”), and distances between positions on RNAP core and positions within σ70 (“core- σ70FRET”). Later the preparation of DNA fragments, sigma σ70, RNAP core, RNAP holoenzyme, and RNA polymerase-promoter open complex (RPo) is discussed. DNA–RNAP FRET permits monitoring of movement of RNAP relative to DNA during promoter escape and elongation and provides information about three-dimensional structures of RPo and RDe. The approach involves two complementary sets of experiments: trailing-edge-FRET experiments, which monitor the distance between a fluorescent probe in RNAP and a fluorescent probe on DNA upstream of RNAP; and leading-edge-FRET experiments, which monitor the distance between a fluorescent probe in RNAP, and a fluorescent probe on DNA downstream of RNAP.