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Related Experiment Videos

Mutations in the occQ operator that decrease OccR-induced DNA bending do not cause constitutive promoter activity.

Reiko Akakura1, Stephen C Winans

  • 1Department of Microbiology, Cornell University, Ithaca, New York 14853, USA.

The Journal of Biological Chemistry
|March 6, 2002
PubMed
Summary
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Octopine perception by OccR, a regulator in Agrobacterium tumefaciens, involves conformational changes. Locking OccR into an active DNA-bound state is insufficient for constitutive activation, indicating octopine induces additional protein changes.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • OccR is a LysR-type transcriptional regulator controlling octopine metabolism in Agrobacterium tumefaciens.
  • Octopine, a plant tumor metabolite, induces conformational changes in DNA-bound OccR, transitioning it from an inactive to an active state.

Purpose of the Study:

  • To investigate the conformational changes of OccR upon DNA binding and octopine induction.
  • To determine if locking OccR into an active conformation is sufficient for constitutive gene regulation.

Main Methods:

  • Gel filtration chromatography to assess OccR oligomerization in solution.
  • Gel shift assays to determine OccR oligomerization state when bound to DNA.
  • Site-directed mutagenesis of the OccR-binding site to create conformationally locked mutants.

Related Experiment Videos

Main Results:

  • OccR exists as a dimer in solution and a tetramer when bound to DNA.
  • Mutations locking OccR into a long footprint/high bend conformation blocked octopine activation.
  • Mutations locking OccR into a short footprint/low bend conformation did not lead to constitutive activation and sometimes interfered with octopine response.

Conclusions:

  • A short footprint conformation is insufficient for constitutive activation of the octopine catabolism operon.
  • Octopine likely induces at least one additional conformational change in OccR beyond DNA binding and bending to achieve full activation.