STRUCTURAL FEATURES CRITICAL TO THE ACTIVITY OF AN ECDYSONE RECEPTOR-BINDING SITE
STRUCTURAL FEATURES CRITICAL TO THE ACTIVITY OF AN ECDYSONE RECEPTOR-BINDING SITE
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DOI：
10.1016/0965-1748(93)90088-a
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发表时间：
1993-01-01
影响因子：
3.8
通讯作者：
LEPESANT, JA
中科院分区：
文献类型：
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作者：
ANTONIEWSKI, C;LAVAL, M;LEPESANT, JA
以黑腹果蝇（Drosophilamelanogaster）hsp 27（hsp 27 EcRE）和Fbp 1（DEcRE）基因中的蜕皮激素反应元件为探针，采用凝胶位移法研究了蜕皮激素受体（EcR）与其同源DNA反应元件的相互作用。EcR的来源是从三龄晚期幼虫脂肪体中提取的核提取物。hsp 27和D EcRE在15 bp区域上的12个位置处共享序列相似性，包括由单个插入核苷酸分隔的两个五聚体半位点组成的不完全回文结构。我们已经证明，一个短的寡核苷酸含有这个11 bp的不完全回文的hsp 27 EcRE和三个侧翼bp的每一侧是一个有效的EcR结合位点。突变分析证实了这两个半位点的完整性以及它们的1bp间距对于蜕皮激素受体的结合是至关重要的。D EcRE表现为比hsp 27 EcRE弱得多的EcR结合位点，但单个bp取代足以赋予其与hsp 27 EcRE的结合能力。这些结果使我们提出序列PuG（G/T）T（C/G）A（N）TG（C/A）（C/A）（C/t）Py作为先前提出的EcRE共有序列的修订版本。
Two ecdysone-response elements from the hsp27 (hsp27 EcRE) and the Fbp 1 (D EcRE) genes of Drosophila melanogaster were used as probes in a gel shift assay to investigate the interactions of the ecdysone receptor (EcR) with its cognate DNA response element. The source of EcR was a nuclear extract from the late third-larval instar fat body. The hsp27 and D EcREs share a sequence similarity at 12 positions over a 15bp region including an imperfect palindromic structure consisting of two pentamer half-sites separated by a single intervening nucleotide. We have shown that a short oligonucleotide containing this 11bp imperfect palindrome of the hsp27 EcRE and three flanking bp on each side is an efficient EcR binding site. Mutational analysis confirms that the integrity of both these half-sites as well as their 1bp spacing are critical for binding of the ecdysone receptor. The D EcRE behaved as a much weaker EcR binding site than the hsp27 EcRE but a single bp substitution was sufficient to confer upon it a binding capacity equivalent to that of the hsp27 EcRE. These results have led us to propose the sequence PuG(G/T)T(C/G)A(N)TG(C/A)(C/A)(C/t)Py as a revised version of a previously proposed EcRE consensus sequence.