Involvement of the σ N DNA‐binding domain in open complex formation

σ N (σ 54 ) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniae σ N mutants, altered in the carboxy DNA‐binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator‐dependent transcription. The mutant holoenzymes have altered activator‐dependent interactions with promoter sequences that normally become melted. Activator‐dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator‐independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I σ N , suggesting different defects. The DNA‐binding domain substitutions are within σ N sequences previously shown to be buried upon formation of the wild‐type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the σ N –core and σ N –DNA interfaces has occurred to change holoenzyme activity. Core‐binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of σ N lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator‐dependent transcription, formed a stable activator‐independent holoenzyme promoter complex on premelted DNA templates.