Transcription of a U6 Small Nuclear RNA Gene

9808214本课题的Kunkel实验旨在探索人小核RNA （snRNA）基因启动子在培养的人细胞中的激活机制。这项工作的主要重点是人类U6基因（尽管其他snRNA基因将在一个项目中进行研究）。脊椎动物U6基因的转录是由RNA聚合酶III进行的，但启动子是由pol II基因中使用的一些DNA控制元件构建的。此外，U6转录复合体部分由pol II和pol III启动子之间共享的因子组成。例如，人类U6基因的远端区域位于转录起始位点上游150 bp以上，包含一个与pol ii转录的snRNA基因（如U1和U2基因）位于相同位置的共识八聚体基序，该元件由纯化的Oct-1因子结合。此外，在许多脊椎动物snRNA基因增强子中常见的SPH基序与人类U6远端启动子中的八聚体基序相邻。本课题的目标是：(1)与人U6远端区相互作用的人sph结合因子（hSBF/ZNF143）的分子表征。使用编码该转录因子的cDNA克隆，将使用瞬时转染和体外转录分析研究负责转录刺激和核定位的蛋白质区域。(2)采用类似的技术，比较hSBF/ZNF143对pol iii转录的U6启动子和pol ii转录的人类U2启动子的转录激活。(3) U6转录复合体中与远端区域结合因子hSBF/ZNF143和Oct-1相互作用的蛋白的鉴定。蛋白质-蛋白质结合将通过使用每种蛋白质特异性抗血清的免疫选择，以及化学和光激活交联剂的使用来研究。小核rna （snRNAs）是真核细胞基因表达的基本途径中的关键成分。此外，snRNA基因是快速生长细胞中最活跃的基因之一。本项目的实验结果将为高表达基因的转录机制提供重要信息。本研究的一个或多个目标将作为研究实验室中至少一名研究生和3-10名本科生培训的基础。

9808214 Kunkel Experiments in this project are designed to explore the mechanism of activation of human small nuclear RNA (snRNA) gene promoters in cultured human cells. The primary focus of this work is on the human U6 gene (although other snRNA genes will be investigated in one project). The transcription of vertebrate U6 genes is carried out by RNA polymerase III, but the promoter is constructed of a number of DNA control elements used in pol II genes. Furthermore, the U6 transcription complex is composed, in part, of factors that are shared between pol II and pol III promoters. For example, the distal region of the human U6 gene, located greater than 150 bp upstream of the transcriptional start site, contains a consensus octamer motif found in the same location as in the pol II-transcribed snRNA genes (e.g., U1 and U2 genes), and this element is bound by purified Oct-1 factor. In addition, a SPH motif that is common to many vertebrate snRNA gene enhancers is adjacent to the octamer motif in the human U6 distal promoter. The objectives of this project are: (1) the molecular characterization of human SPH-binding factor (hSBF/ZNF143), which interacts with the human U6 distal region. Using a cDNA clone encoding this transcription factor, will be investigated regions of the protein that are responsible for transcriptional stimulation and nuclear localization using transient transfection and in vitro transcription assays. (2) A comparison of the transcriptional activation by hSBF/ZNF143 of the pol III-transcribed U6 promoter and the pol II-transcribed human U2 promoter, using similar techniques. (3) The identification of protein(s) in the U6 transcription complex that interact with distal region binding factors, hSBF/ZNF143 and Oct-1. Protein-protein binding will be investigated by immunoselection using antisera specific for each protein, and the use of chemical and photoactivatable cross-linking agents. Small nuclear (snRNAs) are key components in fundamental pathways of gen e expression in eukaryotic cells. Furthermore, snRNA genes are among the most active ones in rapidly growing cells. Results from experiments in this project will provide important information regarding the mechanism of transcription of highly expressed genes. One or more objectives of this research will serve as the basis for the training of at least one graduate student and of 3-10 undergraduate students in the research laboratory.