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.
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 UspDBD(DeltaA)-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.
Faculty of Chemistry, Department of Biochemistry, Wroclaw University of Technology, Wrocław, Poland.
Tomasz
T
Krusiński
Marta
M
Wietrzych
Iwona
I
Grad
Andrzej
A
Ozyhar
Piotr
P
Dobryszycki
D000818:Animals; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000502:physiology; D005454:Fluorescence Polarization / Q000379:methods; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000099190:MAP-Kinase-Activated Kinase 2
D004247:DNA; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D011987:Receptors, Steroid; D014157:Transcription Factors; D000099190:MAP-Kinase-Activated Kinase 2; C066030:USP protein, Drosophila; C055469:ecdysone receptor