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Analysis of trp repressor-operator interaction by filter binding.

L S Klig, I P Crawford, C Yanofsky

    Nucleic Acids Research
    |July 10, 1987
    PubMed
    Summary
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    A new filter binding assay accurately measures trp repressor binding to operator DNA. This method reveals how salt concentration affects binding and dissociation, offering insights into gene regulation.

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • The trp repressor regulates gene expression by binding to operator DNA.
    • Understanding the kinetics of repressor-operator interactions is crucial for deciphering gene regulation mechanisms.

    Purpose of the Study:

    • To develop a simplified filter binding assay for quantifying trp repressor-operator DNA interactions.
    • To determine the dissociation constants and binding stoichiometry of the trp repressor-operator complex.
    • To investigate the influence of corepressors and analogs on repressor-operator binding dynamics.

    Main Methods:

    • Development of a filter binding assay optimized for salt concentration and type.
    • Quantification of trp repressor binding to operator DNA using the developed assay.

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  • Determination of dissociation constants (Kd) for repressor-operator and repressor-corepressor interactions.
  • Analysis of repressor-operator dissociation rates in the presence of L-tryptophan and analogs.

    • Main Results:

    • The assay accurately measures specific trp repressor binding to operator DNA.
    • The dissociation constant (Kd) for the repressor-operator complex was determined to be 2.6 x 10(-9) M.
    • Approximately 1.34 repressor dimers bind per operator DNA molecule.
    • The dissociation constant for the repressor-corepressor (L-tryptophan) complex in the presence of operator DNA is 2.5 x 10(-5) M.
    • Dissociation rates are influenced by tryptophan analog concentrations, suggesting corepressor release as an initial step.

    Conclusions:

    • The developed filter binding assay provides a robust and simpler method for studying repressor-operator interactions.
    • Salt concentration is a critical factor in optimizing trp repressor-operator binding assays.
    • Corepressor binding modulates repressor-operator complex stability, with corepressor release potentially initiating dissociation.