Analysis of Usp DNA binding domain targeting reveals critical determinants of the ecdysone receptor complex interaction with the response element.
The steroid hormone, 20-hydroxyecdysone (20E), directs Drosophila metamorphosis via a heterodimeric receptor formed by two members of the nuclear hormone receptors superfamily, the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. Our previous study [Niedziela-Majka, A., Kochman, M., Ozyhar, A. (2000) Eur. J. Biochem. 267, 507-519] on EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) suggested that UspDBD may act as a specific anchor that preferentially binds the 5' half-site of the pseudo-palindromic response element from the hsp27 gene promoter and thus locates the heterocomplex in the defined orientation. Here, we analyzed in detail the determinants of the UspDBD interaction with the hsp27 element. The roles of individual amino acids in the putative DNA recognition alpha helix and the roles of the base pairs of the UspDBD target sequence have been probed by site-directed mutagenesis. The results show how the hsp27 element specifies UspDBD binding and thus the polar assembly of the UspDBD/EcRDBD heterocomplex. It is suggested how possible nucleotide deviations within the 5' half-site of the element may be used for the fine-tuning of the 20E-response element specificity and consequently the physiological response.
Grad
I
I
Institute of Organic Chemistry, Biochemistry and Biotechnology, Division of Biochemistry, Wroclaw University of Technology, Poland.
Niedziela-Majka
A
A
Kochman
M
M
Ozyhar
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000502:physiology*; D004441:Ecdysterone / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D008675:Metamorphosis, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011987:Receptors, Steroid / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006360:Heat-Shock Proteins; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C055469:ecdysone receptor; D004441:Ecdysterone