Fluorescence Methods for Studying the Kinetics and Thermodynamics of Transcription Initiation

Abstract

This chapter focuses on transient-state kinetic approaches to elucidate the pathway of transcription initiation. These methods are discussed for T7 RNAP, a 99-kDa single subunit phage polymerase that has been characterized extensively, both structurally and mechanistically. The methods described are general and applicable to the studies of other enzymes and polymerases. Transient-state kinetic experiments are designed to follow the formation and decay of reacting species as a function of time. The concentrations are determined directly by radiometric methods or indirectly through optical changes associated with the formation of intermediates and products. Fluorescence methods are described in this chapter to determine the equilibrium-binding constants, and both fluorescence and radiometric methods to determine the intrinsic rate constants of the transcription initiation pathway. The initial steps of RNAP binding to the promoter DNA and formation of the open complex were measured by the stopped-flow fluorescence method. The steps of nucleotide binding and RNA synthesis were measured by both stopped-flow fluorescence and radiometric rapid chemical-quenched flow methods. The chapter emphasizes on the types of experiments that should be used in conjunction, and the data that should be fit globally by numerical methods.