Nucleic Acids Research, Vol 25, Issue 17 3478-3485, Copyright © 1997 by Oxford University Press
XY Wang, A Kolb, W Cannon and M Buck
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.
ARTICLES
Nucleoprotein complex formation by the enhancer binding protein nifA
Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2BB, UK.
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