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Structural Study on the RNA recognition mechanism by the Drosophila Sex-lethal protein

果蝇性致死蛋白RNA识别机制的结构研究

基本信息

批准号：
08680648
负责人：
MUTO Yutaka
金额：
$ 1.66万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680648/
关键词：
RNA binding protein Sxl protein structural biology NMR X-ray chrystallography U2AF65 ELAV-family HuC protein NMR法ショウジョウバエオールタナティブスプライシング性決定 X線結晶構造解析 U2AF蛋白質安定同位体標識法部位特異的変更 BIA core

项目摘要

The Sex-lethal (Sxl) protein of Drosophila melanogaster regulates alternative splicing of the transformer (tra) mRNA precursor by binding to the tra polypyrimidine tract in the sex determination cascade. The Sxl protein has two tandemly-linked RNA binding domains. As the amino-terminal RBD (RBD1) of the Sxl protein exhibits low sequence homology to the typical RBDs, paticularly at the putative functional residues, it was difficult to unambiguously locate the RNP1 and RNP2 motifs. Therefore, at first, we defined the amino and carboxy-terminal borders of the first RNA-binding domain (RBD1) of the Sxl protein by limited tryptic digestion. By replacement of Phe 166 by Tyr, we constructed a highly soluble mutant, which exhibits the same RNA-binding properties as those of the wild-type. Using this mutant protein, we performed NMR measurements, andelucidated the secondary and tertiary structures of the Sxl RBD1 in solution.Second, we have determined the crystal structure of the complex between the two tandemly-arranged RNA binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNa derived from the tra polypyrimidine tract. The two RNA-binding domains have their beta-sheet platforms facing each other, to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognised by the protein. This result provides the first insight into the mechanism by which a protein binds specifically to a cognate RNA that has no intramolecular base pairs.

黑腹果蝇的性致死蛋白（Sxl）通过与性别决定级联中的tra多聚嘧啶束结合来调节Transformer（tra）mRNA前体的选择性剪接。Sxl蛋白具有两个串联连接的RNA结合结构域。由于Sxl蛋白的氨基末端RBD（RBD 1）表现出与典型RBD的低序列同源性，特别是在推定的功能残基处，因此难以明确地定位RNP1和RNP2基序。因此，首先，我们通过有限的胰蛋白酶消化确定了Sxl蛋白的第一个RNA结合结构域（RBD 1）的氨基和羧基末端边界。通过用Tyr替换Phe 166，我们构建了一个高度可溶的突变体，其表现出与野生型相同的RNA结合特性。利用该突变蛋白，我们进行了核磁共振测量，并在溶液中的Sxl RBD 1的二级和三级结构的lucidated。其次，我们已经确定的复合物的晶体结构之间的两个串联排列的RNA结合结构域的Sxl蛋白和12个核苷酸，单链RNa衍生自反式多聚嘧啶道。两个RNA结合结构域的β折叠平台彼此面对，形成V形裂缝。RNA的特征是在这个裂缝中延伸和结合，其中UGUUUUUU序列被蛋白质特异性识别。这一结果首次揭示了蛋白质与没有分子内碱基对的同源RNA特异性结合的机制。