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Related Experiment Videos

Gel retardation at low pH resolves trp repressor-DNA complexes for quantitative study.

J Carey1

  • 1Department of Biochemistry, Stanford University Medical Center, CA 94305-5307.

Proceedings of the National Academy of Sciences of the United States of America
|February 1, 1988
PubMed
Summary
This summary is machine-generated.

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The Escherichia coli trp repressor binds DNA with high affinity, specifically requiring a single dimer for operator binding. This interaction

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The Escherichia coli trp repressor protein regulates the transcription of tryptophan biosynthetic genes.
  • Understanding the precise mechanisms of DNA binding is crucial for gene regulation studies.

Purpose of the Study:

  • To investigate the affinity and stoichiometry of DNA binding by the Escherichia coli trp repressor.
  • To characterize the conditions affecting trp repressor-operator DNA interactions.

Main Methods:

  • Nondenaturing gel electrophoresis to assess DNA binding and mobility shifts.
  • DNase I-protection analysis (footprinting) to determine binding sites and confirm affinities.
  • In vivo labeling of trp repressor to determine complex stoichiometries.

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Main Results:

  • DNA binding affinity is pH-dependent, with enhanced retardation at lower pH.
  • High-affinity operator binding requires a single trp repressor dimer.
  • Apparent dissociation constant (Kd) for trp repressor-operator interaction is 0.5 nM.
  • Nonspecific DNA binding occurs with approximately 200-fold weaker affinity.

Conclusions:

  • The trp repressor exhibits high-affinity, specific binding to its operator DNA sequence.
  • Stoichiometry of specific binding involves one repressor dimer per operator DNA fragment.
  • Gel electrophoresis and footprinting are effective methods for characterizing protein-DNA interactions.