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Interactions between the trp repressor and its operator sequence as studied by base analogue substitution.

J M Mazzarelli1, S B Rajur, P L Iadarola

  • 1Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 01267.

Biochemistry
|June 30, 1992
PubMed
Summary

Modified trp operator sequences reveal key interactions for repressor binding. The thymine carbonyl at position +4 and adenine N7 at +5 are critical for high-affinity binding, while altered adenine amino groups can enhance binding.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • The trp operator is a DNA sequence that regulates the expression of the trp operon.
  • The trp repressor protein binds to the trp operator to control gene transcription.
  • Understanding the molecular interactions between the trp repressor and operator is crucial for gene regulation studies.

Purpose of the Study:

  • To investigate the role of specific base pair functional groups and structural analogues in trp operator-DNA binding.
  • To determine the contribution of individual functional groups to the affinity and specificity of trp repressor-operator interactions.
  • To elucidate the structural basis for high-affinity binding between the trp repressor and its operator sequence.

Main Methods:

  • Synthesis of modified trp operator sequences incorporating base analogues.

Related Experiment Videos

  • Determination of duplex operator melting temperatures (Tm) to assess stability.
  • Utilizing an alkaline phosphatase protection assay to quantify repressor-operator binding affinity.

    • Main Results:

    • Modified trp operator sequences exhibited Tm values correlating with hydrogen bond numbers.
    • The orientation of amino and carbonyl groups at positions -4/+4 is critical for repressor binding.
    • The thymine carbonyl at +4, thymine methyl at +4, and adenine N7 at +5 are essential for high-affinity binding, contributing significantly to binding free energy.
    • Deletion of adenine amino groups at -4 or +5 increased binding affinity, suggesting enhanced flexibility or hydrophobic interactions.

    Conclusions:

    • Specific functional groups on the trp operator, particularly the thymine carbonyl and adenine N7, are critical determinants of trp repressor binding affinity.
    • The orientation of functional groups involved in major groove hydrogen bonding significantly impacts repressor recognition.
    • Alterations in operator sequence, such as the deletion of non-critical amino groups, can lead to enhanced repressor binding through increased flexibility or hydrophobic contacts.