The DNA-binding domain of the ultraspiracle drives deformation of the response element whereas the DNA-binding domain of the ecdysone receptor is responsible for a slight additional change of the preformed structure.
Ecdysteroids control molting and metamorphosis in insects via a heterodimeric complex of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle protein (Usp). We used fluorescence resonance energy transfer (FRET) to study the topology of the natural pseudopalindromic element from the hsp27 gene (hsp27pal) in complex with the DNA-binding domains of Usp and EcR (UspDBD and EcRDBD, respectively). Steady-state data revealed shortening of the end-to-end distance of the hsp27pal-derived probe. For the 70.8 +/- 0.6 A distance obtained for the UspDBD-complexed DNA a bend of about 23.1 +/- 2.9 degrees was measured. Nearly the same value (23.0 +/- 3.4 degrees) was obtained for the DNA complexed with the UspDBD/EcRDBD heterodimer. The respective bend angles estimated using fluorescence decay measurements were 19.0 +/- 2.1 degrees and 20.9 +/- 3.6 degrees . Thus, the FRET data suggest for the first time that the UspDBD defines the architecture of the UspDBD/EcRDBD heterocomplex due to the significant deformation of the hsp27pal. This suggestion has been further reinforced using gel retardation experiments, which, in conjunction with high-resolution DNase I footprinting, indicate that the main contribution to the observed bend is given by the UspDBD itself, while binding of the EcRDBD molecule brings on a slight additional change of the preformed structure.
Dobryszycki
Piotr
P
Division of Biochemistry, Institute of Organic Chemistry, Biochemistry and Biotechnology, Wrocław University of Technology, Wybrzeze Wyspiańskiego 27, 50-370 Wrocław, Poland.
Grad
Iwona
I
Krusiński
Tomasz
T
Michaluk
Piotr
P
Sawicka
Dorota
D
Kowalska
Agnieszka
A
Orłowski
Marek
M
Jakób
Michał
M
Rymarczyk
Grzegorz
G
Kochman
Marian
M
Ozyhar
Andrzej
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D029721:Drosophila Proteins; D031541:Fluorescence Resonance Energy Transfer; D006360:Heat-Shock Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006360:Heat-Shock Proteins; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C055469:ecdysone receptor; D004247:DNA