Telomerase is an important enzyme with implications in stem cell technology, anti cancer drug discovery and design, and understanding of the mechanisms of aging. Telomerase is a remarkable reverse transcriptase that uses its integral RNA subunit as the template for the synthesis of the DNA located at the 3'end of eukaryotic chromosomes. The product of telomerase activity, telomeric DNA, is a single strand of highly repetitive, guanine-rich DNA that is part of the specialized nucleoprotein complex called the telomere. Telomere maintenance is essential because the telomere guards the chromosome ends from mistaken recognition by the DNA repair machinery and nucleolytic degradation. Several features of telomerase remain mysterious. Completion of the studies proposed will illuminate several features of the secondary structure of the RNA subunit of telomerase. The proposed research will also determine the mechanism of the telomerase catalytic reaction cycle, which will allow a more accurate comparison between the structure of telomerase with a broad range of nucleic acid polymerases as well as ribonucleoprotein complexes. Specifically, the secondary structure of telomerase RNA will be elucidated by a combination of site-specific mutants and high resolution biochemical techniques using Tetrahymena thermophila telomerase RNA (tTR) as a model. The first specific aim will use a novel, single nucleotide resolution footprinting technique called selective 2'-hydroxyl acylation by primer extension (SHAPE) to reveal the secondary structure of tTR in complex with the reverse transcriptase domain of telomerase before any nucleotides have been added to the 3'end of telomeric DNA.
The second aim will use SHAPE to reveal the secondary structure of tTR in complex with the reverse transcriptase domain of telomerase in conformations after tTR has been reverse transcribed into a telomeric DNA repeat. The final specific aim will test a specific set of biochemical techniques to trap telomerase in conformations relative to the processes of nucleotide addition and nucleotide polymerization. SHAPE technology will then be used to reveal the secondary structure of tTR during these processes.