RNA POLYMERASE III TRANSCRIPTION REGULATION MECHANISM

除了作为转录研究的模型系统之外 机制，棘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与 戴隐形眼镜。这是一项继续分子研究的建议。 该制剂的生物学，具体地说，引发和 真核生物5S RNA转录的调控。卡氏棘阿米巴， 是研究真核转录的一个很好的模型 生化方法，以及上次资助期间的重大发现 从逻辑上将句号引入拟议的实验。申请人有 从棘阿米巴中克隆5S RNA基因，并对其组织结构进行分析 结构和顺序。克隆的基因序列与 申请人早些时候测序的正品棘阿米巴5S RNA， 并在体外转录，表明它是一个真实的基因，而不是 假基因。5S RNA转录下调是对 停止棘阿米巴生长和包囊，这是一种反应 棘阿米巴等中大rRNA前体的平行下调 真核细胞。拟议中的实验旨在检验 这两个转录单位的协同调控机制。 提出了三类实验：(1)提纯和 反式作用蛋白因子的特性 转录5S RNA基因。在真核生物中，存在一些争议 关于5S RNA需要多少单独的转录因子 而tRNA引发复合体的形成，因此这将是一个值得研究的课题。 (2)5S RNA转录启动机制的研究。 相对较少的完全同源的体外转录系统存在 用于真核细胞的5S RNA。棘阿米巴系统就是其中之一。这 实验室已经广泛地研究了大rrna的机制。 抄写，并有了关于 启动的基本机制。一个单一的转录因子结合 通过蛋白质-蛋白质直接连接到基因的上游位置 相互作用时，RNA聚合酶I到转录起始点。最近， 另一个实验室的结果表明，一种完全相同的 机制适用于RNA聚合酶III的5S RNA和tRNA转录， 但这必须在其他真核系统中重复进行验证。 建议的研究的目的是确认或反驳这是 真核转录启动的普遍机制。分析 启动子序列，特别是那些被认为与中央相互作用的序列 转录因子是计划的。辅助性疾病的机制 “装配”因素将基本因素加载到其站点上，以及如何(和 If)结合影响DNA的构象，从而刺激各种 启动过程中的步骤是需要研究的主题。(三)研究 5S RNA和rRNA转录的协同调控机制。 这个实验室的早期研究表明，大的rRNA 转录是由聚合酶I的修饰来调节的，因此它不会 更长的时间与与启动子结合的因子相互作用。聚合酶I和 III共享一个亚基，在转录过程中两者都被修改 关闭，因此可以通过共同修改来协调调节。 然而，涉及转录因子水平改变或 还将审查活动以及涉及以下方面的机制 将因子重新定位到非核细胞隔间。

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.