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 mus t make productive contact with sigma(54)-holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation proce ss, we sought to isolate mutants of DctD that had reduced affinities f or sigma(54)-holoenzyme. Mutagenesis was confined to the well-conserve d C3 region of the protein, which is required for coupling ATP hydroly sis to open complex formation in sigma(54)-dependent activators. Mutan t forms of DctD that failed to activate transcription and had substitu tions in the C-terminal half of the C3 region were efficiently cross-l inked to sigma(54) and the beta-subunit of RNA polymerase, suggesting that they bound normally to sigma(54)-holoenzyme. In contrast, some mu tant forms of DctD with amino acid substitutions in the N-terminal hal f of the C3 region had reduced affinities for sigma(54) and the beta-s ubunit in the cross-linking assay. These data suggest that the N-termi nal half of the C3 region of DctD contains a site that may contact sig ma(54)-holoenzyme during open complex formation.