Telomerase is a large, multi-subunit ribonucleoprotein complex that is responsible for replication of telomere DNA found at the physical ends of chromosomes. Telomerase is composed of an RNA component that includes the template for DNA synthesis, a catalytic protein component, and a variety of other proteins. Due to the low abundance of telomerase in most organisms and the difficulties in isolating the intact holoenzyme, its exact protein composition is not yet known, and the roles of identified protein and RNA components in RNP processing, assembly, and function have only been partially characterized. In contrast to the very large RNA component of vertebrate and yeast telomerases, the ciliate Tetrahymena telomerase is 159 nts long. The transcriptionally active macronucleus of Tetrahymena thermophila has ~40,000 telomeres, and thus telomerase is more abundant than in other organisms. This has facilitated biochemical characterization of the Tetrahymena telomerase holoenzyme. Because of the smaller size of its RNA and the recent progress in isolating the components of the holoenzyme, Tetrahymena provides a good model system for structural studies of telomerase. This project will focus on structural characterization of the entire T. thermophila telomerase and its interactions with some of the protein components of telomerase using primarily NMR spectroscopy. Although telomerase reverse transcriptase catalyzes nucleotide addition, the RNA does not simply play a role as a scaffold, but rather is essential for telomerase nucleotide and repeat addition processivity. The specific objectives are: (1) Determine the structure of stem-loop IV and characterize its interactions with the RNA binding domain of telomerase reverse transcriptase, the telomerase assembly protein p65, and the pseudoknot; (2) Determine the structures of the putative pseudoknots of Tetrahymena and yeast and compare their structure and function to the human telomerase pseudoknot; (3) Investigate the structure of the large uncharacterized 'single stranded' circle that contains the template and template adjacent regions and its interactions with stem-loop IV. The information from these domain studies will be used to help determine the structure of the full length telomerase.
This project will provide training to undergraduate and graduate students and postdoctoral fellows in NMR structural biology and biophysics of nucleic acids and nucleic acid-protein complexes. This is an area in which women and minorities have long been underrepresented, and the PI's lab provides a role model for them. Results from these studies will likely end up in Biochemistry textbooks; Voet and Voet Biochemistry(3rd edition) includes 2 of the PI's structures.
