RNA POLYMERASE II ELONGATION COMPLEX

这个项目的长期目标是了解真核生物 细胞在转录延伸水平上调节基因的表达。 真核生物调控许多基因的表达，包括c-myc，c-myb， 通过控制RNA聚合酶II合成c-fos的能力 完成初级成绩单。Burkitt病的染色体重排 淋巴瘤与c-myc调节阻断基因紊乱有关 伸长率。调控转录延伸的生化基础 在动物细胞中的作用是未知的，而RNA聚合酶II的伸长复合体是 定义不明确。目前尚不清楚是否有转录起始因子 在聚合酶离开启动子区域后被整合到复合体中。 这项提议是对哺乳动物rna的详细描述。 聚合酶II延伸复合体。我已经将一个站点描述为 阻止RNA聚合酶II和A因子转录的真核基因 (SII)允许通过本网站的延伸。RNA聚合酶II 延长复合体将由大鼠肝脏普通启动组装而成 不含伸长因素的因素。已经开发出一种方法 体外转录抑制伸长复合体的免疫纯化 使用抗RNA的单抗进行反应。孤立的复合体 当受到SII的挑战时，恢复文本延长。的光谱 与延伸复合体相关的启动因子将是 已确认身份。SII的作用机制和体内作用尚不清楚。 还没有观察到稳定的SII与SII反应复合体的结合。 使用我们的方法，我们应该能够检测到SII与复合体的结合 如果结合有很长的半衰期。如果交互是暂时的，我们 应该能够捕获SiI激活的中间体。这是一个 确定SiI介导的生化基础的重要第一步 转录激活。建筑群的大小将由以下因素决定 核酸酶足迹实验和电子显微镜的存在 不含伸长率因子SII。核糖核酸聚合酶II的形态 将对受阻伸长复合体进行调查。这项工作应该是 有助于我们理解RNA对转录延长的控制 聚合酶II。

The long-term objective of this project is to understand how eukaryotic cells regulate gene expression at the level of transcription elongation. Eukaryotes regulate the expression of many genes including c-myc, c-myb, and c-fos by controlling the ability of RNA polymerase II to synthesize complete primary transcripts. Chromosomal rearrangements in Burkitt's lymphoma are associated with the derangement of c-myc's regulated block to elongation. The biochemical basis for regulating transcription elongation in animal cells is unknown and the RNA polymerase II elongation complex is poorly defined. It is unknown if any transcription initiation factors are incorporated into the complex after polymerase leaves the promoter region. THis proposal is a detailed characterization of the mammalian RNA polymerase II elongation complex. I have characterized a site in a eukaryotic gene that blocks transcription by RNA polymerase II and a factor (SII) that allows elongation through this site. RNA polymerase II elongation complexes will be assembled from rat liver general initiation factors which are free of elongation factors. A method has been developed to immunopurify arrested elongation complexes from in vitro transcription reactions using a monoclonal antibody against RNA. Isolated complexes resume transcript elongation when challenged with SII. The spectrum of initiation factors associated with the elongation complex will be identified. The mechanism of action and in vivo role of SII is unclear. Stable SII binding to an SII-responsive complex has not been observed. With our approach, we should be able to detect SII binding to the complex if binding has a long half-life. If the interaction is transient, we should be able to trap the SII-activated intermediate. This is an important first step in determining the biochemical basis for SII-mediated transcriptional activation. The size of the complex will be determined by nuclease footprinting experiments and electron microscopy in the presence and absence of elongation factor SII. The form of RNA polymerase II in an arrested elongation complex will be investigated. This work should facilitate our understanding of the control of transcript elongation by RNA polymerase II.