Tethering σ⁷⁰ to RNA polymerase reveals high in vivo activity of σ factors and σ⁷⁰-dependent pausing at promoter-distal locations

Abstract

Bacterial σ factors compete for binding to RNA polymerase (RNAP) to control promoter selection, and in some cases interact with RNAP to regulate at least the early stages of transcript elongation. However, the effective concentration of σs in vivo, and the extent to which σ can regulate transcript elongation generally, are unknown. We report that tethering σ⁷⁰ to all RNAP molecules via genetic fusion of rpoD to rpoC (encoding σ⁷⁰ and RNAP's β′ subunit, respectively) yields viable Escherichia coli strains in which alternative σ-factor function is not impaired. β′::σ⁷⁰ RNAP transcribed DNA normally in vitro, but allowed σ⁷⁰-dependent pausing at extended -10-like sequences anywhere in a transcriptional unit. Based on measurement of the effective concentration of tethered σ⁷⁰, we conclude that the effective concentration of σ⁷⁰ in E. coli (i.e., its thermodynamic activity) is close to its bulk concentration. At this level, σ⁷⁰ would be a bona fide elongation factor able to direct transcriptional pausing even after its release from RNAP during promoter escape.

Keywords

• RNA polymerase
• σ factor
• transcriptional regulation
• E. coli
• pausing