Transcription of a U6 Small Nuclear RNA Gene

小行星9808214 本项目旨在探索人小核RNA（small nuclear 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基因的转录由RNA聚合酶III进行，但启动子由pol II基因中使用的许多DNA控制元件构成。 此外，U6转录复合物部分由pol II和pol III启动子之间共享的因子组成。 例如，位于转录起始位点上游大于150 bp的人U6基因的远端区含有在与pol II转录的snRNA基因相同的位置发现的共有八聚体基序（例如，U1和U2基因），并且该元件被纯化的Oct-1因子结合。 此外，SPH基序是常见的许多脊椎动物的snRNA基因增强子是相邻的八聚体基序在人类U6远端启动子。 本课题的主要目的是：（1）研究人SPH结合因子（hSBF/ZNF 143）的分子生物学特性。 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测定研究负责转录刺激和核定位的蛋白质区域。 (2)使用类似的技术比较hSBF/ZNF 143对pol III转录的U6启动子和pol II转录的人U2启动子的转录激活。 (3)U6转录复合物中与远端区结合因子hSBF/ZNF 143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对每种蛋白质具有特异性的抗血清的免疫选择以及使用化学和光活化交联剂来研究。 小核RNA（snRNAs）是真核细胞基因表达的重要组成部分. 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在研究实验室培训至少一名研究生和3-10名本科生的基础。

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.