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Related Experiment Videos

Specificity determinants for the interaction of lambda repressor and P22 repressor dimers

F W Whipple1, N H Kuldell, L A Cheatham

  • 1Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.

Genes & Development
|May 15, 1994
PubMed
Summary

Bacteriophage repressors lambda and P22 bind operator DNA cooperatively. Researchers identified specific amino acid changes in lambda repressor that alter its binding specificity, enabling cooperative binding with P22 repressor.

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Area of Science:

  • Molecular Biology
  • Virology
  • Protein-DNA Interactions

Background:

  • Bacteriophage repressors, such as lambda and P22, regulate gene expression by binding to operator sites on DNA.
  • These repressors form dimers, and these dimers can interact with each other (dimer-dimer interaction) to bind cooperatively to operator DNA.
  • Lambda and P22 repressors share sequence homology in their carboxy-terminal domains, which are crucial for dimer formation and dimer-dimer interactions.

Purpose of the Study:

  • To identify specific amino acid residues responsible for the distinct dimer-dimer interaction specificities between lambda and P22 repressors.
  • To engineer a variant lambda repressor with altered specificity for cooperative binding with P22 repressor.

Main Methods:

  • Site-directed mutagenesis was used to introduce amino acid substitutions into the lambda repressor.

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  • Assays were performed to evaluate the effect of these substitutions on DNA-bound lambda repressor dimer-dimer interactions.
  • Relevant substitutions were introduced into the P22 repressor to assess conserved roles.
  • A lambda repressor variant with switched specificity was constructed and characterized.

    • Main Results:

    • Several amino acid substitutions in lambda repressor were found to disrupt its self-interaction.
    • Some of these substitutions had a similar effect on P22 repressor, indicating conserved residues involved in specificity.
    • A lambda repressor variant with six specific amino acid changes exhibited cooperative binding with P22 repressor but not with wild-type lambda repressor.

    Conclusions:

    • Specific amino acid residues within the carboxy-terminal domains of bacteriophage repressors dictate the specificity of dimer-dimer interactions.
    • The study successfully engineered a lambda repressor variant with switched interaction specificity, demonstrating the plasticity of these protein-protein interfaces.
    • This work provides insights into the molecular basis of protein recognition and specificity in DNA-binding proteins.