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

Bacterial sigma 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 sigmas in vivo, and the extent to which sigma can regulate transcript elongation generally, are unknown. We report that tethering sigma(70) to all RNAP molecules via genetic fusion of rpoD to rpoC (encoding sigma(70) and RNAP's beta' subunit, respectively) yields viable Escherichia coli strains in which alternative sigma-factor function is not impaired. beta':: sigma(70) RNAP transcribed DNA normally in vitro, but allowed 4T-dependent pausing at extended -10-like sequences anywhere in a transcriptional unit. Based on measurement of the effective concentration of tethered sigma(70), we conclude that the effective concentration of sigma(70) in E. coli (i.e., its thermodynamic activity) is close to its bulk concentration. At this level, sigma(70) would be a bona fide elongation factor able to direct transcriptional pausing even after its release from RNAP during promoter escape.