Transcription of the Schizosaccharomyces pombe U2 gene in vivo and in vitro is directed by two essential promoter elements.
Transcription of the Schizosaccharomyces pombe U2 gene in vivo and in vitro is directed by two essential promoter elements.
复制标题
DOI：
10.1093/nar/29.10.2003
复制
发表时间：
2001-05
影响因子：
14.9
通讯作者：
D. Zhou;S. Lobo-Ruppert
中科院分区：
文献类型：
--
作者：
D. Zhou;S. Lobo-Ruppert
与后生动物的小核RNA（snRNA）相比，对单细胞生物中snRNA的合成知之甚少。我们分析了粟酒裂殖酵母U2 snRNA基因在体内和同源体外系统中的转录。缺失和连接子扫描分析表明，粟酒裂殖酵母U2启动子至少包含两个元件：位于-55的spUSE，其功能为激活子，以及位于-26的TATA盒，其对于基础转录是必需的。这些数据表明粟酒裂殖酵母、植物和无脊椎动物snRNA启动子之间具有相似的结构。识别spUSE的因子可以通过DNA酶I足迹法在全细胞提取物中检测到，并且竞争研究表明这些因子的结合与转录活性相关。电泳迁移率变动分析和凝胶过滤色谱法揭示了约200 kDa的spUSE结合活性的天然分子量。分子量为25和65 kDa的两种多肽通过其特异性结合spUSE的能力而纯化。
As compared to the metazoan small nuclear RNAs (snRNAs), relatively little is known about snRNA synthesis in unicellular organisms. We have analyzed the transcription of the Schizosaccharomyces pombe U2 snRNA gene in vivo and in the homologous in vitro system. Deletion and linker-scanning analyses show that the S.pombe U2 promoter contains at least two elements: the spUSE centered at -55, which functions as an activator, and a TATA box at -26, which is essential for basal transcription. These data point to a similar architecture among S.pombe, plant and invertebrate snRNA promoters. Factors recognizing the spUSE can be detected in whole cell extracts by DNase I footprinting and competition studies show that the binding of these factors correlates with transcriptional activity. Electrophoretic mobility shift assays and gel-filtration chromatography revealed a native molecular mass of approximately 200 kDa for the spUSE binding activity. Two polypeptides of molecular masses 25 and 65 kDa were purified by virtue of their ability to specifically bind the spUSE.