NMR Studies of RNA Enzymes

RNA 酶的 NMR 研究

• 批准号: 0111060
• 负责人: Juli Feigon
• 金额: $ 36万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0111060&HistoricalAwards=false
• 关键词: NMR; Studies; RNA; Enzymes

Multidimensional, multinuclear NMR spectroscopy will be used to investigate the folding, cation binding, and structures of (1) the hairpin ribozyme from the tobacco ringspot virus satellite and (2) telomerase RNA domains. The hairpin ribozyme studies are a continuation of previous structural studies of the individual domains A and B, and will focus on the folding pathway and cation interactions of the intact ribozyme. Telomerase is a ribonucleoprotein which is responsible for replication of telomere DNA found at the physical ends of chromosomes. Telomerase activity is generally undetectable in somatic cells, and high levels of telomerase activity are only found in proliferating germ cells and cancer cells. The known components of human telomerase are a reverse transcriptase (hTERT) which catalyzes the addition of the nucleotides, a 451 nucleotide RNA (hTR) which contains the template for the synthesized DNA, and telomerase associated protein 1 (TP1). The proposed secondary structure of the vertebrate telomerase RNAs based on phylogeny consists of four conserved structural domains: the pseudoknot domain, the CR4-CR5 domain, the Box H/ACA domain, and the CR7 domain. This essential core structure appears to be preserved despite extensive sequence and length divergence. The conservation of the core telomerase RNA structure and a variety of functional studies both in vitro and in vivo indicate that the structure of the RNA plays an important role in telomerase function. The structures of essential subdomains of telomerase RNA, specifically in the pseudoknot and box H/ACA domains, will be determined and their interactions with known telomerase binding proteins will be studied. These structural studies should complement the existing information on the molecular biology and biochemistry of these enzymes, and provide new insights into RNA folding, tertiary structure, role of cations in tertiary structure formation, and catalysis. In addition, the telomerase studies will provide basic information on the structure and assembly of the telomerase RNP complex.

多维、多核核磁共振波谱将用于研究 (1) 烟草环斑病毒卫星的发夹核酶和 (2) 端粒酶 RNA 结构域的折叠、阳离子结合和结构。发夹核酶研究是先前单个结构域 A 和 B 结构研究的延续，并将重点关注完整核酶的折叠途径和阳离子相互作用。端粒酶是一种核糖核蛋白，负责染色体物理末端端粒 DNA 的复制。体细胞中端粒酶活性一般检测不到，仅在增殖的生殖细胞和癌细胞中发现高水平的端粒酶活性。人类端粒酶的已知成分是催化核苷酸添加的逆转录酶 (hTERT)、包含合成 DNA 模板的 451 核苷酸 RNA (hTR) 和端粒酶相关蛋白 1 (TP1)。基于系统发育的脊椎动物端粒酶RNA的二级结构由四个保守结构域组成：假结域、CR4-CR5域、Box H/ACA域和CR7域。尽管存在广泛的序列和长度差异，但这种重要的核心结构似乎得到了保留。核心端粒酶RNA结构的保守性以及各种体外和体内功能研究表明RNA的结构在端粒酶功能中发挥着重要作用。将确定端粒酶 RNA 的重要子结构域（特别是假结和盒 H/ACA 结构域）的结构，并研究它们与已知端粒酶结合蛋白的相互作用。这些结构研究应该补充这些酶的分子生物学和生物化学的现有信息，并为 RNA 折叠、三级结构、阳离子在三级结构形成中的作用和催化提供新的见解。此外，端粒酶研究将提供端粒酶RNP复合物的结构和组装的基本信息。