Analysis of Usp DNA binding domain targeting reveals critical determinants of the ecdysone receptor complex interaction with the response element
Analysis of Usp DNA binding domain targeting reveals critical determinants of the ecdysone receptor complex interaction with the response element
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DOI：
10.1046/j.1432-1327.2001.02287.x
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发表时间：
2001-07-01
期刊：
影响因子：
--
通讯作者：
Ozyhar, A
中科院分区：
文献类型：
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作者：
Grad, I;Niedziela-Majka, A;Ozyhar, A
类固醇激素，20-羟基蜕皮酮(20E)，通过核激素受体超家族的两个成员-ECR(ECR)和USP(USP)基因的产物形成的异二聚体受体来指导果蝇变态。我们之前的研究[Niedziela-Majka，A.，Kochman，M.，Ozyhar，A.(2000)EU.J.生物化学。267,507-519]在ECR和USP DNA结合域(分别为EcRDBD和UspDBD)上的结果表明，UspDBD可能作为一个特异的锚点，优先结合来自Hsp27基因启动子的假回文反应元件的5‘半个位点，从而将异源复合体定位在指定的方向上。在这里，我们详细分析了UspDBD与Hsp27元件相互作用的决定因素。通过定点突变，探讨了单个氨基酸在DNA识别或螺旋中的作用以及UspDBD靶序列的碱基对的作用。结果表明，Hsp27元件如何指定UspDBD结合，从而指定UspDBD/EcRDBD异质复合体的极性组装。提示了如何利用该元件5‘半位点内的核苷酸偏差来微调20E反应元件的特异性，从而调节生理反应。
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 or 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.