A quantitative assessment for transcriptional pausing of DNA-dependent RNA polymerases in vitro

Abstract

A simple definition for pause strength (tau i) has been given, quantitatively describing transcriptional pausing of RNA polymerases. It permits derivation of practical assessments, based on single-round transcription reactions, which measure the average time a polymerase stops in vitro at certain sites during transcription elongation. We demonstrate that pause strengths can be determined with high accuracy when transcription elongation is started simultaneously from radioactively labeled and purified ternary complexes and transcripts are separated on sequencing gels. Our concept is exemplified by measuring pause strengths on supercoiled templates in the leader region of the Escherichia coli rrnB operon in the presence and the absence of the transcription factor NusA.