Journal of Fluorescence

, Volume 18, Issue 1, pp 1–10 | Cite as

Equilibrium Analysis of the DNA Binding Domain of the Ultraspiracle Protein Interaction with the Response Element from the hsp27 Gene Promoter—the Application of Molecular Beacon Technology

  • Tomasz Krusiński
  • Marta Wietrzych
  • Iwona Grad
  • Andrzej Ożyhar
  • Piotr Dobryszycki
Original Paper


Ecdysteroids initiate molting and metamorphosis in insects via a receptor which belongs to the superfamily of nuclear receptors. The ecdysone receptor consists of two proteins: the ecdysone receptor (EcR) and the ultraspiracle (Usp). The EcR–Usp dimer conducts transcription through a hsp27 pal response element. Usp acts as an anchor orienting the whole complex on the DNA. The molecular beacon methodology was applied to detect the sequence-specific DNA of a natural hsp27 pal or mutated protein interaction with the DNA binding domain from the Usp. The dissociation constant, K d, of the UspDBD–hsp27 pal complex was determined to be 1.42 ± 0.48 nM, whereas K d for UspDBDΔAhsp27 pal was 6.6 ± 0.5 nM. Mutation of Val-71 for Ala blocks formation of the protein-DNA complex in contrast to Glu-19 mutation for Ala for which K d = 4.31 ± 1.01 nM. The results obtained with the molecular beacon technology are related to those obtained by fluorescence anisotropy titrations.


Ecdysteroid receptor Ultraspiracle Fluorescence anisotropy Molecular beacon Drosophila melanogaster 



DNA binding domain




20-hydroxyecdysone response element consisting of an imperfect palindrome from the promoter region of the Drosophila hsp27 gene


retinoid X receptor


product of the ultraspiracle gene


Usp DNA binding domain


product of the EcR gene


fluorescence resonance energy transfer


electrophoretic mobility shift assay



This work was supported by a grant from the Polish Ministry of Science and Higher Education 3552/P01/2006/31. We thank Wroclaw University of Technology for partial support. We thank Dr. A. Kowalska for the gift of UspDBDV71A protein.


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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Tomasz Krusiński
    • 1
  • Marta Wietrzych
    • 1
  • Iwona Grad
    • 2
  • Andrzej Ożyhar
    • 1
  • Piotr Dobryszycki
    • 1
  1. 1.Faculty of Chemistry, Department of BiochemistryWroclaw University of TechnologyWrocławPoland
  2. 2.Department of Cell BiologyUniversity of GenevaGenevaSwitzerland

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