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Related Experiment Videos

Tryptophan super-repressors with alanine 77 changes

D N Arvidson1, J Pfau, J K Hatt

  • 1California Institute of Biological Research, La Jolla 92037.

The Journal of Biological Chemistry
|February 25, 1993
PubMed
Summary

Mutations in the Escherichia coli Trp repressor protein at alanine 77 enhance its activity. Some mutant repressors show improved DNA binding, suggesting altered protein structure and function in gene regulation.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The Escherichia coli Trp repressor (TrpR) regulates tryptophan biosynthesis genes.
  • L-tryptophan binding induces a conformational change, enabling tight operator DNA binding.
  • Residue alanine 77 is in a flexible region crucial for DNA interaction.

Purpose of the Study:

  • To investigate the functional impact of alanine 77 substitutions in the Trp repressor.
  • To identify mutations that enhance aporepressor or holorepressor activity.

Main Methods:

  • Codon-directed mutagenesis to create 19 Ala77 variants of Trp repressor.
  • Analysis of mutant protein expression levels.
  • Challenge phage assays to assess in vivo repressor activity.

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Main Results:

  • All 19 mutant proteins expressed at wild-type levels.
  • Seven mutants (Cys, Ser, Val, Leu, Thr, Ile, Lys) exhibited enhanced aporepressor activity under low tryptophan conditions.
  • Cys and Ser mutants also functioned as super-holorepressors, showing improved repression with excess tryptophan.

Conclusions:

  • Mutations at Ala77 can create super-aporepressors and super-holorepressors.
  • Enhanced activity correlates with predicted disruptions in aporepressor tertiary structure but not holorepressor structure.
  • Super-aporepressors may exhibit reduced affinity for non-specific DNA.