Binding of the 60-kDa Ro autoantigen to Y RNAs: Evidence for recognition in the major groove of a conserved helix
Binding of the 60-kDa Ro autoantigen to Y RNAs: Evidence for recognition in the major groove of a conserved helix
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DOI：
10.1017/s1355838298971667
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发表时间：
1998-07-01
期刊：
影响因子：
4.5
通讯作者：
Wolin, SL
中科院分区：
文献类型：
--
作者：
Green, CD;Long, KS;Wolin, SL
60-kDa Ro自身抗原通常与称为Y RNA的小细胞质RNA复合。在非洲爪蟾卵母细胞中，Ro蛋白也与一大类折叠不正确的变体5S rRNA前体复合。使用纯化的杆状病毒表达的蛋白质，我们表明，60 kDa的Ro蛋白直接结合到Y RNA和错误折叠的5S rRNA前体。为了了解蛋白质如何识别这两种不同类型的RNA，我们研究了Y RNA序列和结构的特征，这些特征是蛋白质识别所必需的。我们确定了一个截短的Y RNA，是稳定的60 kDa的Ro蛋白结合。在这个39-nt RNA中有一个保守的螺旋，被认为是Ro蛋白的结合位点。这个最小的Y RNA的突变显示，由60 kDa的Ro蛋白的结合需要特定的保守螺旋内的碱基对，一个单一的凸起的核苷酸，破坏螺旋，和一个三个核苷酸凸起的相对链。使用焦碳酸二乙酯的化学探测实验表明，在存在两个凸起的情况下，保守螺旋的大沟可以接近蛋白质侧链。这些数据与Ro蛋白通过结合在RNA的大沟中识别保守螺旋中的特定碱基对的模型一致。此外，硫酸二甲酯用于探测裸露和蛋白质结合的Y RNA的实验显示，Ro蛋白结合后RNA中发生结构改变。
The 60-kDa Ro autoantigen is normally complexed with small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the Ro protein is also complexed with a large class of variant 5S rRNA precursors that are folded incorrectly. Using purified baculovirus-expressed protein, we show that the 60-kDa Ro protein binds directly to both Y RNAs and misfolded 5S rRNA precursors. To understand how the protein recognizes these two distinct classes of RNAs, we investigated the features of Y RNA sequence and structure that are necessary for protein recognition. We identified a truncated Y RNA that is stably bound by the 60-kDa Ro protein. Within this 39-nt RNA is a conserved helix that is proposed to be the binding site for the Ro protein. Mutagenesis of this minimal Y RNA revealed that binding by the 60-kDa Ro protein requires specific base pairs within the conserved helix, a singly bulged nucleotide that disrupts the helix, and a three-nucleotide bulge on the opposing strand. Chemical probing experiments using diethyl pyrocarbonate demonstrated that, in the presence of the two bulges, the major groove of the conserved helix is accessible to protein side chains. These data are consistent with a model in which the Ro protein recognizes specific base pairs in the conserved helix by binding in the major groove of the RNA. Furthermore, experiments in which dimethyl sulfate was used to probe a naked and protein-bound Y RNA revealed that a structural alteration occurs in the RNA upon Ro protein binding.