Bacterial RNA polymerase can retain σ70 throughout transcription

Timothy T Harden,Christopher D Wells,Larry J Friedman,Robert Landick,Ann Hochschild,Jane Kondev,Jeff Gelles

doi:10.1073/pnas.1513899113

Abstract

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 σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ(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 σ(70) retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ(70)-RNAP complex during initiation from the λ PR' 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 σ(70) and the kinetics of σ(70) release from actively transcribing complexes. σ(70) release from mature elongation complexes was slow (0.0038 s(-1)); a substantial subpopulation of elongation complexes retained σ(70) throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ(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 σ(70) during transcription.

Highlights

Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination
To observe the presence of σ70 within promoter complexes and transcription elongation complexes (TECs), we tethered linear DNA molecules labeled with AlexaFluor 488 (AF488) dye and containing the phage λ PR′ promoter to the surface of a glass flow chamber (Fig. 1A)
Formation of promoter complexes was visualized as the appearance in total internal reflection fluorescence microscopy [29] of discrete spots of fluorescence that colocalized with the spots from AF488–DNA (Fig. 1B; t = 0)

Summary

Introduction

Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. TECs with σ70 stably bound have been reported [25], and retention of σ70 by TECs stalled at different positions downstream of promoters has been confirmed in bulk [26, 27] and singlemolecule [28] studies The latter data have been interpreted to support models in which σ70 is stochastically released after promoter escape, but there are no studies directly characterizing release kinetics on actively elongating TECs. To understand the postinitiation roles of σ70, it is essential to identify the conditions under which σ70 is retained by RNAP after promoter escape and to describe the kinetics of its release from actively elongating TECs. Here, we use multiwavelength. Elucidating the dynamics of accessory factor binding to and release from the transcription apparatus is essential to achieving a quantitative understanding of the molecular mechanisms that control transcription in cells

Methods

Results

Conclusion