Transcription of a U6 Small Nuclear RNA Gene

该方案中的9304799个昆克尔实验旨在探索人类小核糖核酸基因启动子的激活机制。这些实验的主要焦点是人类的U6基因。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(转录起始点上游超过150bp)包含一个与PolII转录的SnRNA基因(如U1和U2基因)相同位置的共识八聚体基序，该元件在体外与纯化的Oct-1因子结合。因此，U6远端区域类似于(PolII)SnRNA增强子，它激活转录一到两个数量级。本申请中提出的实验目标是：1)定位人类U6基因远端控制区的功能元件的范围。将通过定点技术制备突变模板，并在体外通过凝胶迁移率改变和DNase I足迹分析以及在体内通过在转染的人细胞中瞬时表达的方式进行分析；2)分离已检测到的与U6远端区域结合的转录因子，并将其与Oct-1蛋白(非oct因子)进行区分；3)鉴定U6转录起始途径中由激活蛋白控制的步骤(S)。纯化的Oct-1和Nonoct因子将用于体外转录实验，在这些实验中，使用不同浓度的Sarkosyl或在快速分离后结合到固定化DNA模板上来区分转录起始复合体。将确定功能转录复合体的相对数量、复合体形成的动力学以及激活蛋白对重新启动的可能的差异影响；以及4)确定Oct-1蛋白和Nonoct因子是否相互作用或独立作用激活U6基因转录。非oct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DNase I足迹分析，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如何在脊椎动物细胞中高水平表达。这类基因产生小核RNA(SnRNAs)，这是所有高等细胞中基因表达的基本途径所必需的。这些研究将剖析这类基因中指导快速RNA合成的一个基因(人类U6SnRNA基因)的DNA序列。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的有效生产。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。***

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. ***