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Related Experiment Videos

Nucleotide-dependent conformational changes in the sigma54-dependent activator DctD.

Ying-Kai Wang1, Sungdae Park, B Tracy Nixon

  • 1Department of Microbiology, University of Georgia, Athens, Georgia, USA

Journal of Bacteriology
|October 4, 2003
PubMed
Summary

Sigma(54)-RNA polymerase activators use ATP hydrolysis for promoter complex formation. Structural changes in DctD during ATP binding, detected via DNase I footprinting, suggest communication via the arginine finger.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Microbiology

Background:

  • Sigma(54)-RNA polymerase holoenzyme requires activators to initiate transcription.
  • Activators couple ATP hydrolysis to the formation of an open promoter complex.
  • DctD is a sigma(54)-dependent activator from Sinorhizobium meliloti.

Purpose of the Study:

  • To investigate the structural changes in DctD during ATP hydrolysis.
  • To determine the role of the arginine finger in nucleotide interaction and signal transduction.
  • To characterize the conformational changes of DctD upon ATP binding.

Main Methods:

  • DNase I footprinting assays with wild-type and mutant DctD proteins.
  • Analysis of altered DNase I footprints in the presence of ATP.

Related Experiment Videos

  • Kinetic studies using fluorescence energy transfer (FRET) with Mant-ATP.

    • Main Results:

    • A constitutively active truncated DctD mutant (DctD(Delta1-142)) showed altered DNase I footprints upon ATP binding.
    • A mutant with a substitution near the arginine finger did not exhibit the altered footprint.
    • Kinetic data indicated multiple conformational changes in DctD following ATP binding.

    Conclusions:

    • Structural changes in DctD during ATP hydrolysis are detectable by DNase I footprinting.
    • The arginine finger likely plays a role in communicating nucleotide interactions and conformational changes.
    • DctD undergoes significant conformational rearrangements upon ATP binding, crucial for its function.