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表达。 真核生物调节许多基因的表达，包括c-myc，c-myb， 通过控制RNA聚合酶II合成 完整的初级成绩单。 Burkitt氏病的染色体重排 淋巴瘤与c-myc调节阻滞的紊乱有关， 伸长率 调节转录延伸的生化基础 在动物细胞中是未知的，RNA聚合酶II延伸复合物是 定义不好。 目前尚不清楚是否有转录起始因子被激活。 在聚合酶离开启动子区域后，将其掺入复合物中。 这一建议是一个详细的哺乳动物RNA的特性 聚合酶II延伸复合物。 我已经描述了一个网站， 一种通过RNA聚合酶II和一种因子阻断转录的真核基因 (SII)允许通过这个部位伸长。 RNA聚合酶II 延伸复合物将从大鼠肝脏一般起始组装 不含伸长因子的因子。 已经开发了一种方法 从体外转录中免疫纯化阻滞的延伸复合物 使用针对RNA的单克隆抗体的反应。 孤立络合物 当用SII攻击时恢复转录物延伸。 的光谱 与延伸复合物相关的起始因子将是 鉴定 SII的作用机制和体内作用尚不清楚。 尚未观察到SII与SII响应性复合物的稳定结合。 通过我们的方法，我们应该能够检测到SII与复合物的结合 如果结合具有较长的半衰期。 如果相互作用是短暂的，我们 应该能捕获SII激活的中间体 这是一 重要的第一步，在确定SII介导的生化基础， 转录激活 建筑群的规模将由 核酸酶足迹实验和电子显微镜在存在 以及缺乏延伸因子SII。 RNA聚合酶II的形式， 将研究阻滞伸长复合物。 这项工作应 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.