Analysis of Usp DNA binding domain targeting reveals critical determinantsof the ecdysone receptor complex interaction with the response element

The steroid hormone, 20-hydroxyecdysone (20E), directs Drosophila metamorph osis via a heterodimeric receptor formed by two members of the nuclear horm one receptors superfamily, the product of the EcR (EcR) and of the ultraspi racle (Usp) genes. Our previous study [Niedziela-Majka, A., Kochman, M., Oz yhar, A. (2000) Eur. J. Biochem. 267, 507-519] on EcR and Usp DNA-binding d omains (EcRDBD and UspDBD, respectively) suggested that UspDBD may act as a specific anchor that preferentially binds the 5' half-site of the pseudo-p alindromic response element from the hsp27 gene promoter and thus locates t he heterocomplex in the defined orientation. Here, we analyzed in detail th e determinants of the UspDBD interaction with the hsp27 element. The roles of individual amino acids in the putative DNA recognition or helix and the roles of the base pairs of the UspDBD target sequence have been probed by s ite-directed mutagenesis. The results show how the hsp27 element specifies UspDBD binding and thus the polar assembly of the UspDBD/EcRDBD heterocompl ex. It is suggested how possible nucleotide deviations within the 5' half-s ite of the element may be used for the fine-tuning of the 20E-response elem ent specificity and consequently the physiological response.