Nucleic Acid Structure and Reactivity

描述(申请人提供)：端粒是真核生物线性染色体的末端，由一种名为端粒酶的特殊RNA蛋白质酶复制。这个项目的长期目标是帮助理解端粒酶的组装、结构、活性和调节，特别是它的RNA成分。由于端粒酶在大多数人类体细胞中是不活跃的，但在大多数肿瘤中是重新激活的，因此对该酶的分析可能有助于癌症化疗药物的发展。端粒酶的关键特征在系统发育上是保守的，因此选择实验系统来进行最深入的分析。 在第一组特定目标中，将在酿酒酵母中确定端粒酶RNA的结构，这将有助于遗传和分子分析。特定的功能，如结合蛋白质亚单位或相对于模板定位活性部位，将被分配给RNA结构元件。 第二组特定的目标涉及纤毛虫的端粒酶，选择它是因为它的简单和生物化学上的易操纵性。P43蛋白亚基是端粒酶全酶组装的伴侣蛋白，其与RNA的结合和功能作用将被详细分析。 由于端粒酶的作用在很大程度上是由与端粒DNA结合的蛋白质调控的，第三个特定目的涉及最近发现的POT1(端粒保护)蛋白，它在包括人类和分裂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核苷酸是由哪些氨基酸识别的，从而了解不同生物的POT1蛋白是如何识别不同的端粒DNA序列的。

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.