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Related Experiment Videos

Nucleoprotein complex formation by the enhancer binding protein nifA

X Y Wang1, A Kolb, W Cannon

  • 1Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2BB, UK.

Nucleic Acids Research
|September 1, 1997
PubMed
Summary

Nitrogen fixation protein NifA binds to upstream activator sequences (UASs), with DNA bending by integration host factor (IHF) facilitating interaction with sigma54 RNA polymerase holoenzyme. This study reveals NifA

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Area of Science:

  • Molecular Biology
  • Microbial Genetics
  • Biochemistry

Background:

  • The nitrogen fixation protein NifA activates transcription via the sigma54 RNA polymerase holoenzyme.
  • NifA binding sites (UASs) are remotely located, requiring DNA bending by integration host factor (IHF) for interaction with the holoenzyme.
  • Understanding these interactions is crucial for deciphering bacterial nitrogen fixation regulation.

Purpose of the Study:

  • To investigate the precise contacts between NifA and the Klebsiella pneumoniae nifH promoter UAS.
  • To determine the role of holoenzyme and IHF in NifA-UAS interactions.
  • To elucidate the DNA conformational changes associated with promoter complex formation.

Main Methods:

  • UV light footprinting on 5-BrdU-substituted DNA.

Related Experiment Videos

  • DNase I footprinting on conventional DNA.
  • Laser UV footprinting on conventional DNA templates.
  • Titration assays to assess binding cooperativity.

    • Main Results:

    • Consensus thymidine residues (5'-TGT) of the UAS are in close proximity to NifA, with differential reactivity observed.
    • NifA binding to the UAS shows weak cooperativity with holoenzyme or IHF.
    • Holoenzyme binding suppresses reactivity of promoter T residues (-14, -15, -16), indicating close contact.
    • Holoenzyme binding induces DNA distortion at -9, requiring N-terminal sigma54 sequences for activation.
    • A distinct footprint signal change at -11 during open complex formation suggests strand separation nucleation near the -12 promoter element.

    Conclusions:

    • NifA directly contacts specific thymidine residues within the UAS.
    • The activation mechanism likely does not rely on simple recruitment of factors.
    • Closed promoter complexes are poised for DNA melting, with strand separation initiating near the -12 promoter element.
    • These findings provide insights into the molecular mechanism of sigma54-dependent transcriptional activation.