Transcription of a U6 Small Nuclear RNA Gene

本项目中的9808214个昆克尔实验旨在探索人小核糖核酸基因启动子在培养的人类细胞中激活的机制。这项工作的主要焦点是人类的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PolII基因中使用的一些DNA控制元件组成的。此外，U6转录复合体部分由PolII和PolIII启动子共享的因子组成。例如，人类U6基因的远端区域位于转录起始点上游超过150bp的位置，包含一个与PolII转录的SnRNA基因(例如U1和U2基因)位于同一位置的共识八聚体基序，该元件与纯化的Oct-1因子结合。此外，在许多脊椎动物的SnRNA基因增强子中，SPH基序与人类U6远端启动子中的八聚体基序相邻。本项目的目标是：(1)与人U6远端区域相互作用的人SPH结合因子(hSBF/ZNF143)的分子特征。利用编码该转录因子的cDNA克隆，将利用瞬时转染和体外转录试验来研究负责转录刺激和核定位的蛋白质区域。(2)用相似的技术比较hSBF/ZNF143对polIII转录的U6启动子和PolII转录的人U2启动子的转录激活作用。(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(S)的鉴定。蛋白质与蛋白质的结合将通过使用针对每种蛋白质的抗血清的免疫选择以及使用化学和可光激活的交联剂来研究。小核糖核酸是真核细胞中基因e表达的基本途径中的关键成分。此外，在快速生长的细胞中，SnRNA基因是最活跃的基因之一。该项目的实验结果将提供有关高表达基因转录机制的重要信息。这项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.