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Related Experiment Videos

Lambda cI repressor mutants altered in transcriptional activation

P Kolkhof1, B Müller-Hill

  • 1Institut für Genetik, Universität zu Köln, Germany.

Journal of Molecular Biology
|September 9, 1994
PubMed
Summary

We investigated lambda cI repressor mutants affecting positive control (pc). One mutant (D38-N) lost activation but retained DNA binding, while others showed altered binding affinities, revealing key residues for repressor function.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Bacteriophage Lambda Research

Background:

  • The lambda cI repressor regulates gene expression in bacteriophage lambda.
  • Positive control (pc) is crucial for efficient gene activation by the cI repressor.
  • Understanding pc mechanisms requires analyzing repressor mutants with altered functions.

Purpose of the Study:

  • To analyze the in vivo functions of three lambda cI repressor mutants defective in positive control.
  • To investigate the impact of specific amino acid substitutions on repressor DNA binding and activation.
  • To elucidate the role of residues at positions 34, 38, and 43 in cI repressor activity.

Main Methods:

  • Constructed a lambda cI repressor expression system for controlled in vivo expression.

Related Experiment Videos

  • Measured PRM promoter activation by wild-type and mutant cI repressors.
  • Studied DNA binding properties of repressors and mutants using various operator constructs in vivo.

    • Main Results:

    • Wild-type cI repressor showed a five-fold activation of the PRM promoter.
    • Two mutants (G43-R, E34-K) repressed the PRM promoter, indicating altered DNA binding.
    • One mutant (D38-N) retained DNA binding but lost activation function, behaving as a true pc mutant.
    • Amino acid substitutions at position 38 (D38-N, D38-Y, D38-F) differentially affected activation and binding.

    Conclusions:

    • Amino acid changes at positions 34 and 43 primarily impact lambda cI repressor binding properties.
    • Hydrophobic residues at position 38 are as functional, or more so, than the wild-type acidic residue for activation.
    • The D38-N mutant provides a model for studying positive control mechanisms independently of DNA binding.