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(Delta A)-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.