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Related Experiment Videos

Direct and indirect readout in mutant Met repressor-operator complexes.

C W Garvie1, S E Phillips

  • 1Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, LS2 9JT, Leeds, UK.

Structure (London, England : 1993)
|September 15, 2000
PubMed
Summary
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The methionine repressor (MetJ) binds DNA operator sequences with varying affinity, influenced by direct contacts and DNA conformation. Even non-contacted bases affect binding stability, revealing complex sequence recognition mechanisms.

Area of Science:

  • Molecular Biology
  • Structural Biology
  • Genetics

Background:

  • The methionine repressor, MetJ, regulates methionine biosynthesis gene transcription by binding to specific DNA sequences called 'metboxes'.
  • Natural operator sequences exhibit variations from the consensus 'metbox' sequence, particularly with multiple binding sites.
  • MetJ demonstrates sensitivity to specific base changes, even at non-contacted DNA sites.

Purpose of the Study:

  • To investigate the structural basis of MetJ's sequence recognition, focusing on its interaction with mutated operator sequences.
  • To elucidate the roles of direct and indirect readout mechanisms in MetJ-DNA binding affinity and specificity.

Main Methods:

  • High-resolution structural analysis of MetJ mutants (Q44K) complexed with consensus and mutated operator DNA sequences.

Related Experiment Videos

  • Comparative analysis of wild-type and mutant MetJ-DNA complex structures to identify differences in DNA-protein interactions and conformation.

    • Main Results:

    • The Q44K MetJ mutant structure bound to DNA is similar to the wild-type, with subtle alterations in DNA backbone conformation and base contacts.
    • Mutant complexes revealed a combination of direct and indirect DNA sequence readout mechanisms.
    • Differences in direct contacts and DNA conformation were observed in the mutant complexes, impacting binding affinity.

    Conclusions:

    • MetJ utilizes strong interactions with the DNA sugar-phosphate backbone to tolerate some operator sequence variation.
    • Reduced binding affinity in mutant complexes is partly due to loss of direct DNA contacts.
    • Unfavorable DNA conformation, caused by specific base-step alterations, can significantly decrease repressor-operator complex stability.