Nucleic Acid Structure and Reactivity

核酸结构和反应性

• 批准号: 6542339
• 负责人: THOMAS ROBERT CECH
• 金额: $ 25.3万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-08-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6542339
• 关键词: Ciliophora; DNA binding protein; RNA; Saccharomyces cerevisiae; aminoacid; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme structure; fungal genetics; gel mobility shift assay; immunoprecipitation; northern blottings; nucleic acid structure; protein binding; protein structure function; telomerase; telomere; western blottings

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.

描述（由申请人提供）：端粒，线性真核染色体的末端，由一种称为端粒酶的专用RNA蛋白酶复制。该项目的长期目标是有助于理解端粒酶的组装，结构，活动和调节，并特别强调其RNA成分。由于端粒酶在大多数人类体细胞中都是不活跃的，但在大多数肿瘤中都重新激活，因此对该酶的分析可能有助于癌症化学疗法的发展。端粒酶的关键特征是系统发育保守的，因此选择实验系统以允许最敏锐的分析。 在第一组特定目的中，将在酿酒酵母中确定端粒酶RNA的结构，该结构促进遗传和分子分析。特定功能，例如结合蛋白质亚基或将活动位点相对于模板的定位，将分配给RNA结构元素。 第二组特定的目的涉及纤毛效应拟南芥的远处酶，因为它的简单性和生化障碍性而选择。一种称为p43的蛋白质亚基是用于组装端粒酶全酶的推定伴侣，将详细分析其RNA结合和功能作用。 由于端粒酶作用在很大程度上由与端粒DNA结合的蛋白质调节，因此第三个特定目的涉及最近发现的POT1（保护端粒）蛋白，该蛋白在包括人类和裂变酵母在内的真核生物中广泛保守。这里的目的是确定哪些端粒DNA核苷酸是识别的，通过哪种氨基酸，可以理解来自不同生物体的POT1蛋白如何识别不同的端粒DNA序列。