title: Bacterial RNA polymerase can retain σ70 throughout transcription.
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.
authors.affiliation: Department of Physics, Brandeis University, Waltham, MA 02454; Department of Biochemistry, Brandeis University, Waltham, MA 02454;
authors.forename: Timothy T
authors.initials: TT
authors.lastname: Harden
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115;
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Wells
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454;
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Friedman
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706; Department of Bacteriology, University of Wisconsin, Madison, WI 53706.
authors.forename: Robert
authors.initials: R
authors.lastname: Landick
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115;
authors.forename: Ann
authors.initials: A
authors.lastname: Hochschild
authors.affiliation: Department of Physics, Brandeis University, Waltham, MA 02454; kondev@brandeis.edu gelles@brandeis.edu.
authors.forename: Jane
authors.initials: J
authors.lastname: Kondev
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454; kondev@brandeis.edu gelles@brandeis.edu.
authors.forename: Jeff
authors.identifier: http://orcid.org/0000-0001-7910-3421
authors.initials: J
authors.lastname: Gelles
mesh_terms: D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D007834:Lasers; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D038761:Photobleaching; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000378:metabolism*; D013698:Templates, Genetic; D013997:Time Factors; D061805:Transcription Elongation, Genetic; D014158:Transcription, Genetic
chemical_list: D009711:Nucleotides; D012333:RNA, Messenger; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases
keywords: CoSMoS; elongation complex; sigma factor; single-molecule fluorescence; transcription regulation