AraC mutations that suppress inactivating mutations in the C-terminal domain of the RpoA subunit ofEscherichia coliRNA polymerase

Abstract

AbstractInE. coli, transcriptional activation is often mediated by the C-terminal domain of RpoA, the α subunit of RNA polymerase. Mutations that prevent activation of the arabinose PBADpromoter are clustered in a small region of the α–CTD domain around K271. To determine the target(s) of RpoA in the PBADpromoter, we have isolated suppressors ofrpoAα–CTD mutations. The suppressors map to the N-terminal domain of AraC, the main transcriptional regulator ofaragene expression. No mutation was found in the large DNA regulatory region betweenaraCand PBAD, suggesting that, in this system, RpoA does not activate transcription through its direct DNA binding. One class ofaraCmutations result in substitutions in the core of the N-terminal domain suggesting that they may affect its conformation. Another class of suppressors define genetically a domain that potentially interacts with the C-terminal domain of RpoA. Surprisingly, inrpoA+strains lacking CRP, thearaCmutations largely restore arabinose gene expression, suggesting that they somehow strengthen the AraC:α–CTD interaction. Thus, the N-terminal domain of AraC exhibits at least three activities: dimerization, arabinose binding and transcriptional activation via RpoA.ImportanceGene expression is most often controlled at the level of transcription by regulators that interact with RNA polymerase. The C-terminal domain ofEscherichia coliRpoA is attached to the core enzyme by a flexible linker and serves as a hub that interacts with many regulators and even with DNA sites to activate transcription. Mutations in a RpoA subdomain interfere with activation of the main arabinose promoter by AraC, the regulator that either activates or represses expression of the arabinose operons. We define here genetically the target of RpoA in the main arabinose promoter. Suppressors of most RpoA mutations map to the N-terminal domain of AraC that promotes its dimerization and binds to arabinose, the inducer. Thus, our results identify a third function for this AraC domain. Some suppressors define a potential binding site for RpoA, while others, at internal residues, probably affect the conformation of the AraC domain.