title: Functionality versus strength -- has functional selection taken place in the case of the ecdysteroid receptor response element?.
abstract: Nuclear receptors are ligand-dependent transcription factors responsible for controlling differentiation, growth and development of higher eukaryotes. Three amino acids within the recognition alpha-helix of the DNA-binding domain of the nuclear receptors constitute the so-called "P-box" which determines response element specificity. In the ultraspiracle (Usp) protein, which together with EcR forms the heterodimeric ecdysone receptor, the P-box residues are E19, G20 and G23. Substitution of E19, the most characteristic amino acid for estrogen receptor-like P-boxes, with alanine showed that the mutation did not appreciably alter the affinity of the wild-type Usp DNA-binding domain (UspDBD(WT)) for a probe containing natural ecdysone response element (hsp27(wt)). Since in many cases E19 contacts a G/C base pair in position -4, which is absent in hsp27(wt), we analysed the interaction of UspDBD(WT), E19A and other P-box region mutants with the hsp27(wt) derivative which contains a G/C instead of an T/A base pair in position -4. UspDBD(WT) exhibited higher affinity for this element than for hsp27(wt). Moreover, a different interaction pattern of P-box region mutants was also observed. Thus we conclude that the E19 residue of UspDBD is not involved in any hsp27(wt) sequence-discerning contacts. However, substitution of the hsp27(wt) T/A base pair in position -4 with G/C generates target sequence with distinct functional characteristics and possibly with a new specificity. These results could serve as a basis for understanding the role of the presence of a T/A or G/C base-pair in the position -4 in the two types of ecdysone response elements found in nature.
authors.affiliation: Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw University of Technology, Wrocław, Poland.
authors.forename: Iwona
authors.initials: I
authors.lastname: Grad
authors.forename: Marian
authors.initials: M
authors.lastname: Kochman
authors.forename: Andrzej
authors.initials: A
authors.lastname: Ozyhar
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism; D004441:Ecdysterone / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000502:physiology*; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D011987:Receptors, Steroid; D011994:Recombinant Proteins; C034808:ecdysteroid receptor; D004441:Ecdysterone