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

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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.