NMR Studies of RNA Enzymes

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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.