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Related Experiment Videos

Distances between DNA and ATP binding sites in the TyrR-DNA complex.

W H Sawyer1, R Y Chan, J F Eccleston

  • 1The Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Australia 3052. w.sawyer@biochemistry.unimelb.edu.au

Biochemistry
|May 10, 2000
PubMed
Summary

The regulatory protein TyrR in Escherichia coli binds ATP away from its DNA-binding domain. This distance suggests ATP binding induces significant conformational changes, affecting TyrR

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Escherichia coli regulatory protein TyrR controls aromatic amino acid biosynthesis and transport.
  • TyrR functions as a homodimer, binding DNA and utilizing ATP.
  • Understanding TyrR's interaction with DNA and ATP is crucial for elucidating gene regulation.

Purpose of the Study:

  • To determine the spatial relationship between the ATP binding site and DNA in the TyrR-DNA complex.
  • To investigate the structural basis for ATP's allosteric effects on TyrR function.

Main Methods:

  • Utilized fluorescence resonance energy transfer (FRET) to measure distances.
  • Synthesized DNA oligomers with fluorescein labels at specific positions.
  • Used rhodamine-labeled ATP as the FRET acceptor.

Related Experiment Videos

  • Determined TyrR-ATP binding affinity using fluorescence anisotropy titrations.

    • Main Results:

    • The ATP binding site is located 40-45 Å from the nearest DNA contact point.
    • The ATP binding site is spatially distant from the helix-turn-helix DNA binding motif.
    • ATP binding increases TyrR's affinity for DNA by 4-5 fold.

    Conclusions:

    • ATP binding to TyrR induces significant allosteric conformational changes.
    • These conformational changes are responsible for enhanced DNA binding and corepressor interaction.
    • The results provide structural insights into TyrR-mediated gene regulation.