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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

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Abstract

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 UspDBDΔA–hsp27 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.

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Abbreviations

DBD:

DNA binding domain

20E:

20-hydroxyecdysone

hsp27 pal :

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

RXR:

retinoid X receptor

Usp:

product of the ultraspiracle gene

UspDBD:

Usp DNA binding domain

EcR:

product of the EcR gene

FRET:

fluorescence resonance energy transfer

EMSA:

electrophoretic mobility shift assay

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Acknowledgments

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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Author notes

  1. Iwona Grad

    Present address: Department of Cell Biology, University of Geneva, Geneva, Switzerland

Authors and Affiliations

  1. Faculty of Chemistry, Department of Biochemistry, Wroclaw University of Technology, Wybrzeże Wyspiańskiego 27, 50-370, Wrocław, Poland

    Tomasz Krusiński, Marta Wietrzych, Andrzej Ożyhar & Piotr Dobryszycki

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  1. Tomasz Krusiński
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  2. Marta Wietrzych
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  3. Iwona Grad
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Correspondence to Piotr Dobryszycki.

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Krusiński, T., Wietrzych, M., Grad, I. et al. 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. J Fluoresc 18, 1–10 (2008). https://doi.org/10.1007/s10895-007-0285-y

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