Mapping the Escherichia coli transcription elongation complex with exonuclease III.

Abstract

RNA polymerase interactions with the nucleic acids control every step of the transcription cycle. These contacts mediate RNA polymerase recruitment to promoters, induce pausing during RNA chain synthesis, and control transcription termination. These interactions are dissected using footprinting assays, in which a bound protein protects nucleic acids from the digestion by nucleases or modification by chemical probes. Exonuclease III is frequently employed to study protein-DNA interactions owing to relatively simple procedures and low background. Exonuclease III has been used to determine RNA polymerase position in transcription initiation and elongation complexes and to infer the translocation register of the enzyme. In this chapter, we describe probing the location and the conformation of transcription elongation complexes formed by walking of the RNA polymerase along an immobilized template.