RNA POLYMERASE III TRANSCRIPTION REGULATION MECHANISM

RNA聚合酶III转录调控机制

• 批准号: 3273525
• 负责人: Marvin R. Paule
• 金额: $ 12.85万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-02-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3273525
• 关键词: Acanthamoeba; DNA directed RNA polymerase; RNA biosynthesis; cell free system; conformation; developmental genetics; enzyme mechanism; gene deletion mutation; gene expression; genetic manipulation; genetic mapping; genetic promoter element; genetic transcription; molecular cloning; nucleic acid sequence; nucleic acid structure; point mutation; pseudogenes; ribosomal RNA; transcription factor; transfer RNA; translation factor

In addition to its serving as a model system for studies of transcription mechanisms, Acanthamoeba is the causative agent of amoebic keratitis, a serious and currently difficult to treat corneal infection associated with contact lens wear. This is a proposal to continue studies of the molecular biology of this agent, specifically, the mechanism of initiation and regulation of eukaryotic 5S RNA transcription. Acanthamoeba castellanii, is an excellent model for the study of eukaryotic transcription using biochemical approaches, and significant findings during the last funding period lead logically into the proposed experiments. The applicant has cloned the 5S RNA gene from Acanthamoeba, and analyzed its organizational structure and sequence. The cloned gene sequence matches the sequence of authentic Acanthamoeba 5S RNA, which the applicant had sequenced earlier, and is transcribed in vitro, showing that it is a true gene and not a pseudogene. 5S RNA transcription is down regulated in response to cessation of growth and encystment of Acanthamoeba, a response which parallels down regulation of large rRNA precursor in Acanthamoeba nd other eukaryotic cells. The experiments proposed are aimed at an examination of the mechanism of coordinate regulation of these two transcription units. Three categories of experiments are proposed: (1) Purification and characterization of the trans-acting protein factors required for transcription of the 5S RNA gene. In eukaryotes, there is some controversy concerning how many separate transcription factors are needed for 5S RNA and tRNA initiation complex formation, so this will be a topic of study. (2) Studies of the mechanism of initiation of 5S RNA transcription. Relatively few completely homologous in vitro transcription systems exist for eukaryotic 5S RNA. The Acanthamoeba system is one of them. This laboratory has extensively studied the mechanism of large rRNA transcription, and have made important discoveries concerning the fundamental mechanisms of initiation. A single transcription factor binds to an upstream site on the gene and directs, by protein-protein interaction, RNA polymerase I to the transcription start site. Recently, results from another laboratory have suggested that an exactly homologous mechanism applies to 5S RNA and tRNA transcription by RNA polymerase III, but this must be repeated in other eukaryotic systems for verification. The aim of the studies proposed is to confirm or disprove that this is the universal mechanism of eukaryotic transcription initiation. Analysis of promoter sequences, especially those believed to interact with the central transcription factor are planned. The mechanism by which the ancillary "assembly" factors load the fundamental factor onto its site, and how (and if) binding affects the conformation of the DNA so as to stimulate various steps in the initiation process are topics for study. (3) Study the mechanism of coordinate regulation of 5S RNA and rRNA transcription. Earlier studies from this laboratory demonstrated that large rRNA transcription is regulated by modification of polymerase I so that it no longer interacts with the factor bound to the promoter. Polymerases I and III share a subunit which is modified in both during transcriptional shutdown, so coordinate regulation by co-modification is possible. However, mechanisms involving alteration of transcription factor levels or activities will also be examined, as well as mechanisms involving relocation of factors into non nuclear cellular compartments.

除了作为转录研究的模型系统外， 机制，阿米巴是阿米巴角膜炎的病原体， 严重且目前难以治疗的角膜感染， 接触透镜磨损。 这是一个继续研究分子的建议， 这种药剂的生物学，特别是启动机制， 真核生物5S RNA转录的调控。 卡氏阿米巴， 是研究真核生物转录的极好模型， 生物化学方法，以及在上一次资助期间的重大发现 在逻辑上，这一时期将导致拟议的实验。 申请人已经 克隆了阿米巴5S RNA基因，并对其进行了组织结构分析 结构和顺序。 克隆的基因序列与 真实的阿米巴5S RNA，申请人之前已经测序， 并在体外转录，这表明它是一个真正的基因，而不是一个 假基因 5S RNA转录下调， 停止生长和包囊的阿米巴，一种反应， 在阿米巴和其他阿米巴中大rRNA前体的下调平行 真核细胞 提出的实验旨在检查 这两个转录单位的协调调节机制。 提出了三类实验：（1）纯化和 所需的反式作用蛋白质因子的表征 5S RNA基因的转录。 在真核生物中， 关于5S RNA需要多少个独立的转录因子 和tRNA起始复合物的形成，所以这将是一个研究课题。 (2)5S RNA转录起始机制的研究。 完全同源的体外转录系统相对较少 真核生物的5S RNA。 阿米巴系统就是其中之一。 这 一个实验室已经广泛研究了大rRNA的机制， 并取得了重要的发现， 启动的基本机制。 单个转录因子结合 到基因的上游，并通过蛋白质-蛋白质 RNA聚合酶I与转录起始位点的相互作用。 最近， 另一个实验室的结果表明， 机制适用于5S RNA和RNA聚合酶III的tRNA转录， 但这必须在其他真核系统中重复以进行验证。 所提出的研究的目的是证实或反驳这是 真核生物转录起始的普遍机制。 分析 启动子序列，特别是那些被认为与中央 转录因子是有计划的。 辅助机构的运作机制 “组装”因子将基本因子加载到其站点上，以及如何（以及 如果）结合影响DNA的构象，从而刺激各种 启动过程的步骤是研究的主题。 (3)研究 5S RNA和rRNA转录的协调调节机制。 该实验室的早期研究表明， 转录通过聚合酶I的修饰来调节，因此它不会 与结合到启动子上的因子的相互作用时间更长。 聚合酶I和 III共有一个亚基，在转录过程中， 关闭，因此通过共同修饰的协调调节是可能的。 然而，涉及转录因子水平改变或 还将审查各项活动，以及涉及 将因子重新定位到非核细胞区室中。