Nucleic Acid Structure and Reactivity

描述（由申请人提供）：端粒是线性真核染色体的末端，由一种称为端粒酶的特殊 RNA 蛋白酶进行复制。该项目的长期目标是促进对端粒酶的组装、结构、活性和调节的理解，特别是其 RNA 成分。由于端粒酶在大多数人体细胞中不活跃，但在大多数肿瘤中重新激活，因此对该酶的分析可能有助于癌症化疗药物的开发。端粒酶的关键特征在系统发育上是保守的，因此选择实验系统以进行最深入的分析。 在第一组具体目标中，将确定酿酒酵母中端粒酶 RNA 的结构，这有助于遗传和分子分析。特定功能，例如结合蛋白质亚基或相对于模板定位活性位点，将被分配给 RNA 结构元件。 第二组具体目标涉及纤毛虫 Euplotes aediculcitus 的端粒酶，之所以选择它是因为其简单性和生化易处理性。一种名为 p43 的蛋白质亚基是端粒酶全酶组装的假定伴侣，我们将详细分析其 RNA 结合和功能作用。 由于端粒酶的作用在很大程度上是由与端粒 DNA 结合的蛋白质调节的，因此第三个具体目标涉及最近发现的 Pot1（端粒保护）蛋白质，该蛋白质在包括人类和裂殖酵母在内的真核生物中广泛保守。这里的目标是确定哪些端粒 DNA 核苷酸被哪些氨基酸识别，从而了解来自不同生物体的 Pot1 蛋白如何识别不同的端粒 DNA 序列。

DESCRIPTION (provided by applicant): Telomeres , the ends of linear eukaryotic chromosomes, are replicated by a specialized RNA-protein enzyme called telomerase. The long-term goal of this project is to contribute to the understanding of the assembly, structure, activity and regulation of telomerase, with special emphasis on its RNA component. Because telomerase is inactive in most human somatic cells but reactivated in most tumors, analysis of this enzyme may contribute to the development of cancer chemotherapeutics. Key features of telomerase are phylogenetically conserved, so experimental systems are chosen to allow the most incisive analysis. In the first set of specific aims, the structure of the telomerase RNA will be determined in the yeast Saccharomyces cerevisiae, which facilitates genetic as well as molecular analysis. Specific functions, such as binding a protein subunit or positioning the active site relative to the template, will be assigned to RNA structure elements. The second set of specific aims involves the telornerase of the ciliate Euplotes aediculcitus, chosen because of its simplicity and biochemical tractability. A protein subunit called p43 is a putative chaperone for assembly of the telomerase holoenzyme, and its RNA-binding and functional roles will be analyzed in detail. Because telomerase action is regulated in large part by proteins that bind to the telomeric DNA, the third specific aim concerns the recently discovered Pot1 (Protection Of Telomeres) protein, which is widely conserved among eukaryotes including humans and fission yeast. The goal here is to determine which telomeric DNA nucleotides are recognized by which amino acids, providing an understanding of how the Pot1 proteins from different organisms recognize different telomeric DNA sequences.