A non-native C-terminal extension of the beta' subunit compromises RNA polymerase and Rho functions

Escherichia coli RfaH abrogates Rho-mediated polarity in lipopolysaccharide core biosynthesis operons, and Delta rfaH 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) beta' 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 omega chaperone subunit, restores rpoC397* growth at 42 degrees C but has no effect on SDS sensitivity. Our results suggest that the C397* tail traps the omega subunit in an inhibitory state through direct contacts and hinders Rho-dependent termination through long-range interactions. We propose that the dynamic and hypervariable beta'center dot omega module controls RNA synthesis in response to niche-specific signals.

Automated summary

Escherichia coli RfaH plays a role in abolishing Rho-mediated polarity in lipopolysaccharide core biosynthesis operons. A temperature-sensitive mutant rpoC397* was identified as a suppressor of SDS sensitivity, with 46 C-terminal residues of the RNA polymerase beta' subunit replaced by 23 residues carrying a net positive charge. The rpoC397* mutant mimics RfaH's resistance to Rho and exhibits similar responses to pause signals and Nus factors, while still binding to Rho and Nus factors in vivo.