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Related Experiment Videos

Designed hyperstable Lac repressor.DNA loop topologies suggest alternative loop geometries.

R A Mehta1, J D Kahn

  • 1Program in Molecular and Cell Biology, University of Maryland, College Park, MD 20742-2021, USA.

Journal of Molecular Biology
|November 11, 1999
PubMed
Summary
This summary is machine-generated.

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Lac repressor (LacI) protein forms DNA loops for gene repression. Engineered DNA loops with bent sequences and LacI operators showed hyperstable structures, offering a method to stabilize protein-DNA complexes.

Area of Science:

  • Molecular Biology
  • Biophysics
  • Structural Biology

Background:

  • Lac repressor (LacI) is crucial for transcriptional repression via DNA looping.
  • Understanding LacI DNA loop conformations is key to gene regulation mechanisms.

Purpose of the Study:

  • To characterize DNA loops formed by LacI on engineered DNA constructs with phased A-tract bends.
  • To investigate the stability and topology of different LacI-DNA loop conformations.

Main Methods:

  • Mobility shift assays
  • DNA footprinting
  • DNA cyclization and topology analysis
  • Competition experiments

Main Results:

  • Engineered DNA loops exhibited increased stability, particularly the SL/WT construct with half-lives of days.

Related Experiment Videos

  • DNA cyclization yielded altered topoisomers, with WA constructs showing relaxed and positive topoisomers, and SL/WT showing relaxed and slightly negative supercoiling.
  • Results suggest a U-shaped loop geometry around an extended LacI tetramer is most stable, differing from some proposed models.

    • Conclusions:

    • Bent DNA constructs can stabilize LacI-DNA loops, providing a general approach for protein-DNA complex stabilization.
    • Loop topology, including induced supercoiling, influences LacI binding and potentially subsequent protein assembly.
    • The study reconciles LacI's preference for negative supercoiling with its ability to induce positive supercoiling in loops.