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Tn10 tet operator mutations affecting Tet repressor recognition.

A Wissmann, I Meier, L V Wray

    Nucleic Acids Research
    |May 27, 1986
    PubMed
    Summary
    This summary is machine-generated.

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    Altering single base pairs in the Tn10 tet operator affects Tet repressor recognition. Specific GC base pairs at positions 2, 6, and 8 are crucial for this interaction, with decreasing importance in that order.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Biochemistry

    Background:

    • The Tn10 tetracycline resistance (tet) operon is regulated by the Tet repressor protein binding to specific operator sequences.
    • Understanding the precise molecular interactions between the Tet repressor and its operator is crucial for deciphering gene regulation mechanisms.

    Purpose of the Study:

    • To investigate the impact of single base pair alterations within the Tn10 tet operator on Tet repressor binding affinity.
    • To identify specific nucleotides within the tet operator essential for Tet repressor recognition.

    Main Methods:

    • In vivo repressor titration assays using a lacZ reporter system to measure operator-repressor interactions.
    • In vitro dissociation rate determinations to quantify the binding kinetics of Tet repressor to altered operator sequences.

    Related Experiment Videos

  • Analysis of synthetic operator sequences to assess binding affinities.

    • Main Results:

    • Separating the tandem O1 and O2 operators resulted in a two-fold difference in Tet repressor affinity.
    • The Tet repressor exhibited higher affinity for the non-palindromic O2 wild-type operator compared to palindromic sequences.
    • Mutational analysis revealed that GC base pairs at positions 2, 6, and 8 of the tet operator are critical for Tet repressor recognition, with their importance decreasing in the order 2 > 6 > 8.
    • Transitions at position 7 had less impact on recognition than transversions.

    Conclusions:

    • The study quantitatively correlates in vivo and in vitro data, confirming the roles of specific base pairs in Tet repressor-operator binding.
    • The findings provide detailed insights into the molecular basis of Tet repressor recognition of the Tn10 operator sequence.