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，线性真核细胞染色体的末端，由称为端粒酶的专门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，活性和调节，特别强调其RNA组分。由于端粒酶在大多数人体细胞中是无活性的，但在大多数肿瘤中被重新激活，因此对这种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遗传学上是保守的，因此选择实验系统进行最深入的分析。 在第一组具体目标中，端粒酶RNA的结构将在酿酒酵母中确定，这有助于遗传和分子分析。特定的功能，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。 第二组具体的目标涉及纤毛类动物Euplotes aediculcitus的端粒酶，选择它是因为它的简单性和生物化学的易处理性。一种称为p43的蛋白质亚基是端粒酶全酶组装的假定伴侣，其RNA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结合的蛋白质调节的，第三个特定的目标涉及最近发现的Pot1（端粒保护）蛋白，该蛋白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。