Zhou D, Lobo-Ruppert S M
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 844 BBRB, 845 19th Street South, Birmingham, AL 35294, USA.
Nucleic Acids Res. 2001 May 15;29(10):2003-11. doi: 10.1093/nar/29.10.2003.
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.
与后生动物的小核RNA（snRNA）相比，关于单细胞生物中snRNA合成的了解相对较少。我们已经在体内和同源体外系统中分析了粟酒裂殖酵母U2 snRNA基因的转录。缺失和接头扫描分析表明，粟酒裂殖酵母U2启动子至少包含两个元件：位于-55处的spUSE，其作为激活剂发挥作用；以及位于-26处的TATA框，它对于基础转录至关重要。这些数据表明粟酒裂殖酵母、植物和无脊椎动物的snRNA启动子具有相似的结构。通过DNase I足迹法可在全细胞提取物中检测到识别spUSE的因子，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显示，spUSE结合活性的天然分子量约为200 kDa。通过其特异性结合spUSE的能力纯化出了分子量分别为25 kDa和65 kDa的两种多肽。
