Involvement of the sigma(N) DNA-binding domain in open complex formation

sigma(N) (sigma(54)) RNA polymerase holoenzyme closed complexes isomerize t o open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella p neumoniae sigma(N) mutants, altered in the carboxy DNA-binding domain (F354 A/F355A, F402A, F403A and F402A/F403A), that fail in activator-dependent tr anscription. The mutant holoenzymes have altered activator-dependent intera ctions with promoter sequences that normally become melted. Activator depen dent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtaine d in an assay of activator-independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex forma tion but did for deleted region I sigma(N), suggesting different defects. T he DNA-binding domain substitutions are within sigma(N) sequences previousl y shown to be buried upon formation of the wild-type holoenzyme or closed c omplex, suggesting that, in the mutants, alteration of the sigma(N)-core an d sigma(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 sigma(N) lacking the major core binding determina nt was able to assemble into holoenzyme and, although unable to support act ivator-dependent transcription, formed a stable activator-independent holoe nzyme promoter complex on premelted DNA templates.