Escherichia coli RfaH abrogates Rho‐mediated polarity in lipopolysaccharide core biosynthesis operons, and ΔrfaH cells are hypersensitive to antibiotics, bile salts, and detergents. Selection for rfaH suppressors that restore growth on SDS identified a temperature‐sensitive mutant in which 46 C‐terminal residues of the RNA polymerase (RNAP) β’ subunit are replaced with 23 residues carrying a net positive charge. Based on similarity to rpoC397, which confers a temperature‐sensitive phenotype and resistance to bacteriophages, we named this mutant rpoC397*. We show that SDS resistance depends on a single nonpolar residue within the C397* tail, whereas basic residues are dispensable. In line with its mimicry of RfaH, C397* RNAP is resistant to Rho but responds to pause signals, NusA, and NusG in vitro similarly to the wild‐type enzyme and binds to Rho and Nus factors in vivo. Strikingly, the deletion of rpoZ, which encodes the ω “chaperone” subunit, restores rpoC397* growth at 42°C but has no effect on SDS sensitivity. Our results suggest that the C397* tail traps the ω subunit in an inhibitory state through direct contacts and hinders Rho‐dependent termination through long‐range interactions. We propose that the dynamic and hypervariable β’•ω module controls RNA synthesis in response to niche‐specific signals.

Method is available in the published article.