A conserved region in the sigma(54)-dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

Rhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between sigma(54)-RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with sigma(54)-holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for sigma(54)-holoenzyme. Mutagenesis was confined to the well-conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in sigma(54)-dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C-terminal half of the C3 region were efficiently cross-linked to sigma(54) and the beta-subunit of RNA polymerase, suggesting that they bound normally to sigma(54)-holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N-terminal half of the C3 region had reduced affinities for sigma(54) and the beta-subunit in the cross-linking assay. These data suggest that the N-terminal half of the C3 region of DctD contains a site that may contact sigma(54)-holoenzyme during open complex formation.