Transcription of a U6 Small Nuclear RNA Gene

U6 小核 RNA 基因的转录

• 批准号: 9304799
• 负责人: Gary Kunkel
• 金额: $ 27.28万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9304799&HistoricalAwards=false
• 关键词: Transcription; U6; Small; Nuclear; RNA

9304799 Kunkel Experiments in this proposal are designed to explore the mechanism of activation of human small RNA gene promoters. The primary focus of these experiments is on the human U6 gene. The transcription of vertebrate U6 genes is carried out by RNA polymerase III but the promoter is constructed of a number of DNA 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 (more 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 vitro. Therefore, the U6 distal region is similar to the (pol II)snRNA enhancer that activates transcription by one to two orders of magnitude. The objectives of experiments proposed in this application are: 1) to map the extent of functional elements in the human U6 gene distal control region. Mutant templates will be prepared by site-directed techniques, and analyzed in vitro by gel mobility shift and DNase I footprinting assays and in vivo by transient expression in transfected human cells; 2) to isolate a transcription factor that binds to the U6 distal region whose presence has been detected already and is distinct from Oct-1 protein (Nonoct factor); 3) to characterize the step(s) in the U6 transcription initiation pathway that are controlled by activator proteins. Purified Oct-1 and Nonoct factors will be used for in vitro transcription experiments in which transcription initiation complexes are distinguished using various concentrations of Sarkosyl or after rapid separation while attached to bead-immobilized DNA templates. The relative numbers of functional transcription complexes, kinetics of complex formation and possible differential effects on reinitiation by the activator proteins will be determined; and 4) to determine whether Oct-1 protein and Nonoct factor interact or act independently to activate U6 gene transcription. The spacing between the Nonoct and octamer protein binding sites will be altered systematically, and the effect on transcriptional activation determined. Furthermore, using a DNase I footprinting assay, binding of these two factors to DNA will be investigated to determine whether cooperativity exists. %%% This research is designed to elucidate how an important class of genes are expressed at a high level in vertebrate cells. This class of genes produces the small nuclear RNAs (snRNAs) that are essential for fundamental pathways of gene expression in all higher cells. The studies will dissect the DNA sequences for one gene in this class (human U6 snRNA gene) that direct rapid RNA synthesis. Then the investigators will identify important proteins that bind to these DNA sequences and determine how these proteins stimulate efficient production of U6 snRNA. These studies will provide important information on basic mechanisms that direct how specific genes are highly expressed. ***

9304799本方案的Kunkel实验旨在探索人小RNA基因启动子的激活机制。这些实验的主要焦点是人类U6基因。脊椎动物U6基因的转录是由RNA聚合酶III完成的，但启动子是由pol II基因中使用的一些DNA元件构成的。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（转录起始位点上游超过150 bp）包含一个共识的八聚体基序，与pol ii转录的snRNA基因（如U1和U2基因）位于相同的位置，该元件在体外被纯化的Oct-1因子结合。因此，U6远端区域与激活转录的（pol II）snRNA增强子相似，其激活程度为一到两个数量级。本应用程序提出的实验目标是：1)绘制人类U6基因远端控制区功能元件的范围。突变模板将通过位点定向技术制备，并通过凝胶迁移转移和dna酶I足迹分析体外和体内通过转染的人细胞中的瞬时表达进行分析；2)分离一个与U6远端区结合的转录因子，该转录因子的存在已经被检测到，并且与Oct-1蛋白（Nonoct因子）不同；3)表征由激活蛋白控制的U6转录起始途径中的步骤。纯化的Oct-1和Nonoct因子将用于体外转录实验，其中转录起始复合物使用不同浓度的萨科齐或快速分离后连接到头部固定的DNA模板来区分。将确定功能性转录复合物的相对数量、复合物形成的动力学以及激活蛋白对再起始可能产生的不同影响；4)确定Oct-1蛋白与Nonoct因子是相互作用还是单独作用激活U6基因转录。Nonoct和八聚体蛋白结合位点之间的间距将被系统地改变，并确定对转录激活的影响。此外，使用DNA酶I足迹测定，将研究这两个因子与DNA的结合，以确定是否存在协同作用。这项研究旨在阐明一类重要的基因是如何在脊椎动物细胞中高水平表达的。这类基因产生的小核rna （snrna）是所有高等细胞中基因表达的基本途径所必需的。该研究将剖析这类基因（人类U6 snRNA基因）的DNA序列，该基因指导快速RNA合成。然后，研究人员将确定与这些DNA序列结合的重要蛋白质，并确定这些蛋白质如何刺激U6 snRNA的有效产生。这些研究将为指导特定基因如何高度表达的基本机制提供重要信息。＊＊＊