Bacterial RNA polymerase can retain σ 70 throughout transcription

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 113; no. 3; pp. 602 - 607
• Main Authors: Harden, Timothy T., Wells, Christopher D., Friedman, Larry J., Landick, Robert, Hochschild, Ann, Kondev, Jane, Gelles, Jeff
• Format: Journal Article
• Language: English
• Published: 19.01.2016
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1513899113

Significance In all kingdoms of life, gene transcription is not carried out by RNA polymerase enzymes alone. Instead, the behavior of RNA polymerases during transcription initiation, elongation, and termination is regulated by accessory proteins that bind to the polymerase molecule. Bacterial σ proteins are historically thought of as transcription initiation factors primarily involved in promoter recognition. Here, we use light microscopy to directly observe the behavior of individual fluorescently labeled σ 70 subunits during transcript elongation by Escherichia coli RNA polymerase. We show that σ 70 can be retained on an RNA polymerase molecule throughout transcription and alters polymerase behavior during transcript elongation. 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 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 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.