FACTORS INVOLVED IN TRANSCRIPTION BY RNA POLYMERASE II

RNA 聚合酶 II 转录涉及的因素

• 批准号: 2177928
• 负责人: David H Price
• 金额: $ 23.38万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-01-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177928
• 关键词: DNA directed RNA polymerase; Drosophilidae; Escherichia coli; RNA biosynthesis; binding proteins; enzyme mechanism; gel electrophoresis; gene expression; genetic manipulation; genetic promoter element; genetic transcription; ion exchange chromatography; messenger RNA; nucleic acid sequence; polymerase chain reaction; protein reconstitution; tissue /cell culture; transcription factor

The goal of this proposed project is to come to a better understanding of the elongation phase of transcription by RNA polymerase II. The formation and maintenance of elongation complexes which are competent to synthesize mRNAs will be investigated. One of the major goals of the work is to characterize two recently identified factors that seem to be involved in controlling the formation of such complexes. One of these factors is tightly coupled to the preinitiation complex and forces the polymerase into an elongation mode in which only short transcripts can be formed. The second factor acts after initiation and allows the polymerase to proceed to the 3' end of the transcribed gene. An in vitro approach utilizing Drosophila Kc cell nuclear extracts will be taken for all of the proposed studies. DNA templates immobilized to paramagnetic beads will be used to manipulate transcription complexes formed on physiological promoters. Dissection of the processes leading to the formation of mature elongation complexes will be accomplished by changing the factors present in the reactions following magnetic concentration of the complexes. An examination of the properties of mature elongation complexes will determine how stably the elongation factors interact with the complex and if elongation complexes formed by initiation from different promoters have different properties. Another goal is to determine the biochemical details of the function of DmS-II, a Drosophila RNA polymerase II elongation factor. These studies will be aided by the availability of large amount of the intact protein and domains of the protein which have been produced in E. coli from the cDNA. The clear involvement of elongation control in the expression of numerous oncogenes and viruses including HIV, demands that the biochemical details of the process be elucidated so that rational approaches to controlling the diseased state may be made. The proposed studies should shed light on the mechanisms controlling elongation and, furthermore, will allow the formation of a more coherent picture of the complete transcription cycle.

这个拟议项目的目标是更好地理解 RNA聚合酶II的转录伸长阶段。 形成 以及维护延伸络合物的合成能力 将调查mRNA。 工作的主要目标之一是 描述了两个最近发现的因素，这些因素似乎参与了 控制这种复合物的形成。 这些因素之一是 紧密耦合与预启用复合物并迫使聚合酶进入 伸长模式只能形成短成绩单。 这 启动后的第二个因素行动，并允许聚合酶继续进行 转录基因的3'末端。 利用的体外方法 果蝇KC细胞核提取物将用于所有提议的 研究。 固定在顺磁珠上的DNA模板将用于 在生理启动子上形成的操纵转录复合物。 解剖过程导致成熟伸长的形成 复合物将通过更改中存在的因素来实现 复合物的磁浓度后的反应。 一个 检查成熟伸长率复合物的性能将确定 伸长因子与复合物相互作用以及if 由不同启动子启动形成的伸长络合物具有 不同的属性。 另一个目标是确定生化细节 DMS-II的功能，果蝇RNA聚合酶II伸长率 因素。 这些研究将通过大量的可用性来帮助 在 来自cDNA的大肠杆菌。 伸长控制的明显参与 包括HIV在内的许多癌基因和病毒的表达，要求 阐明该过程的生化细节，以便理性 可以控制患病状态的方法。 提议 研究应阐明控制伸长的机制和 此外，将允许形成更连贯的图片 完整的转录周期。