Nucleoprotein complex formation by the enhancer binding protein nifA.
The nitrogen fixation protein NifA is a member of the protein family activating transcription by the alternative eubacterial sigmaN (sigma54) RNA polymerase holoenzyme. Binding sites for NifA, upstream activator sequences (UASs), are remotely located. Interaction between holoenzyme bound in a closed promoter complex and NiFA is facilitated by bending of the intervening DNA by integration host factor (IHF). We have examined NifA contact with the Klebsiella pneumoniae nifH promoter UAS 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 consensus thymidine residues of the UAS motif 5'-TGT are in close proximity to NifA. Reactivity suggests that each UAS thymidine is not structurally equivalent. Titration of NifA binding to the UAS in the presence or absence of the closed promoter complex indicates that the interaction of NifA with the UAS is not strongly co-operative with holoenzyme or IHF, a result supportive of an activation mechanism not reliant upon simple recruitment of factors to the promoter. Laser footprints demonstrated that holoenzyme suppressed reactivity of promoter consensus -14, -15 and -16 T residues, indicating close contact. Binding of holoenzyme resulted in a specific increase in 5-BrdU reactivity at -9 within the holoenzyme binding site, likely reflecting DNA distortion. Enhanced -9 reactivity required sigmaNN-terminal sequences that are necessary for activation. Since T-9 is melted in open complexes the closed complex appears poised for melting. Open promoter complex formation was accompanied by a distinct change in laser footprint signal at -11, consistent with the view that nucleation of strand separation occurs within or close to the -12 promoter element.
Wang
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Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2BB, UK.
Kolb
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Cannon
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Buck
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D016948:Azotobacter vinelandii / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001973:Bromodeoxyuridine / Q000378:metabolism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I; D034982:Integration Host Factors; D007711:Klebsiella pneumoniae / Q000737:chemistry; D009698:Nucleoproteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; C061338:NifA protein, Bacteria; D009698:Nucleoproteins; D012808:Sigma Factor; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D003850:Deoxyribonuclease I; D001973:Bromodeoxyuridine