AraC Functional Suppressors of Mutations in the C-Terminal Domain of the RpoA Subunit of the Escherichia coli RNA Polymerase

In <i>E. coli</i>, transcriptional activation is often mediated by the C-terminal domain of RpoA, the α subunit of RNA polymerase. Random mutations that prevent activation of the arabinose P_BAD promoter are clustered in the RpoA C-terminal domain (α-CTD). We have isolated functional suppressors of <i>rpoA</i> α-CTD mutations that map to <i>araC</i>, the main transcriptional regulator of <i>ara</i> genes, or to the P_BAD promoter. No mutation was found in the DNA regulatory region between <i>araC</i> and P_BAD. Most suppressors that improve P_BAD transcription are localized to the N-terminal domain of AraC. One class of <i>araC</i> mutations generates substitutions in the core of the N-terminal domain, suggesting that they affect its conformation. Other suppressors localize to the flexible N-terminal arm of AraC. Some, but not all, suppressors confer an arabinose constitutive phenotype. Suppression by both classes of <i>araC</i> mutations requires the α-CTD to stimulate expression from P_BAD. Surprisingly, in <i>rpoA</i>⁺ strains lacking Crp, the cAMP receptor protein, these <i>araC</i> mutations largely restore arabinose gene expression and can essentially bypass Crp activation. Thus, the N-terminal domain of AraC exhibits at least three distinct activities: dimerization, arabinose binding, and transcriptional activation. Finally, one mutation maps to the AraC C-terminal domain and can synergize with AraC mutations in the N-terminal domain.