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Trp holorepressor-trp operator interaction studied by protein distribution analysis.

P V Haydock, R L Somerville

    Biochemical and Biophysical Research Communications
    |March 30, 1984
    PubMed
    Summary

    The Trp repressor protein from E. coli changes shape when it binds L-tryptophan, allowing it to specifically attach to DNA operator sites. Some repressor protein remains inactive for DNA binding.

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    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • The Trp repressor protein in Escherichia coli regulates tryptophan biosynthesis.
    • This repressor controls gene expression by binding to specific DNA operator sequences.
    • Understanding repressor-DNA interactions is crucial for gene regulation studies.

    Purpose of the Study:

    • To investigate the conformational changes of the Trp repressor upon L-tryptophan binding.
    • To characterize the binding kinetics and stoichiometry of the Trp holorepressor-operator complex.
    • To identify any inactive fractions of the Trp repressor in operator binding.

    Main Methods:

    • Protein distribution analysis was employed to assess repressor activity.
    • Equilibrium dissociation constant (Kd) was determined for the holorepressor-operator interaction.

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  • Binding stoichiometry was analyzed at varying molar ratios of protein to DNA.

    • Main Results:

    • Trp repressor undergoes a conformational change upon L-tryptophan interaction, forming an active holorepressor.
    • The holorepressor binds specifically to double-stranded DNA operator targets.
    • A significant fraction of Trp repressor was found to be inert in operator binding.
    • The equilibrium dissociation constant for the Trp holorepressor-operator interaction is 6.7 nM.
    • The complex formation involves a 1:1 stoichiometry of holorepressor to operator.

    Conclusions:

    • L-tryptophan binding induces a specific DNA-binding conformation in the Trp repressor.
    • The Trp repressor system exhibits high specificity and affinity for its operator targets.
    • The presence of an inactive repressor fraction warrants further investigation into its biological significance.