NMR Studies of RNA Enzymes

多维多核NMR光谱将用于研究（1）烟草环斑病毒卫星的发夹状核酶和（2）端粒酶RNA结构域的折叠、阳离子结合和结构。发夹状核酶的研究是对单个结构域A和B的结构研究的延续，并将集中于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，负责染色体末端端粒DNA的复制。端粒酶活性通常在体细胞中检测不到，并且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。人端粒酶的已知组分是催化核苷酸添加的逆转录酶（hTERT）、含有合成DNA的模板的451个核苷酸的RNA（hTR）和端粒酶相关蛋白1（TP 1）。脊椎动物端粒酶RNA的二级结构由四个保守的结构域组成：假结结构域、CR 4-CR 5结构域、Box H/ACA结构域和CR 7结构域。这种基本的核心结构似乎被保留，尽管广泛的序列和长度的分歧。核心端粒酶RNA结构的保守性和各种体内外功能研究表明，RNA的结构在端粒酶功能中起着重要作用。端粒酶RNA的基本亚结构域的结构，特别是在假结和盒H/ACA结构域，将被确定，并与已知的端粒酶结合蛋白的相互作用将被研究。这些结构的研究应补充现有的信息，这些酶的分子生物学和生物化学，并提供新的见解RNA折叠，三级结构，阳离子在三级结构形成中的作用，和催化。此外，端粒酶的研究将提供端粒酶RNP复合物的结构和组装的基础信息。

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.