Bacterial RNA polymerase can retain σ70 throughout transcription

Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated sigma factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary s factor in Escherichia coli, sigma(70), can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of sigma(70) retention have been limited to complexes halted during early elongation. Here, we used multiwave-length single-molecule fluorescence-colocalization microscopy to observe the sigma(70)-RNAP complex during initiation from the lambda P-R' promoter and throughout the elongation of a long (> 2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound sigma(70) and the kinetics of sigma(70) release from actively transcribing complexes. sigma(70) release from mature elongation complexes was slow (0.0038 s(-1)); a substantial subpopulation of elongation complexes retained sigma(70) throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing sigma(70) manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of sigma(70) during transcription.