NUCLEOPROTEIN COMPLEX-FORMATION BY THE ENHANCER-BINDING PROTEIN NIFA

The nitrogen fixation protein NifA is a member of the protein family a ctivating transcription by the alternative eubacterial sigma(N) (sigma (54)) RNA polymerase holoenzyme, Binding sites for NifA, upstream acti vator sequences (UASs), are remotely located, Interaction between holo enzyme bound in a closed promoter complex and NIFA is facilitated by b ending of the intervening DNA by integration host factor (IHF), We hav e examined NifA contact with the Klebsiella pneumoniae nifH promoter U AS in the presence and absence of holoenzyme and IHF, Footprints with UV light were made on 5-BrdU-substituted DNA and DNase I and laser UV footprints on conventional DNA templates, Results establish that the c onsensus thymidine residues of the UAS motif 5'-TGT are in close proxi mity to NifA, Reactivity suggests that each UAS thymidine is not struc turally equivalent, Titration of NifA binding to the UAS in the presen ce or absence of the closed promoter complex indicates that the intera ction of NifA with the UAS is not strongly co-operative with holoenzym e or IHF, a result supportive of an activation mechanism not reliant u pon simple recruitment of factors to the promoter, Laser footprints de monstrated that holo-enzyme binding site, likely reflecting DNA distor tion. Enhanced -9 reactivity required sigma(N) N-terminal sequences th at are necessary for activation, Since T-9 is melted in open complexes the closed complex appears poised for molting, Open promoter complex formation was accompanied by a distinct change in laser footprint sign al at -11, consistent with the view that nucleation of strand separati on occurs within or close to the -12 promoter element.