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Interrogation of Flexibility in Sequential Uridine Residues in Stem IV of Tetrahymena TERC (2010)

Undergraduates: Ishita Das, Vijay Sekaran


Faculty Advisor: Michael Jarstfer
Department: Biology


Telomeres are DNA sequences at the ends of chromosomes that protect them from degradation, fusion, and recombination. In somatic cells, telomeres shorten with each cell division, eventually signaling for cell death. However, cancer cells can up-regulate the enzyme telomerase, a ribonucleoprotein complex involved in the addition of six nucleotide repeats to DNA at the end of the chromosomes lengthening the telomeres and allowing cells to live longer and proliferate. Crucial to telomerase activity is the telomerase RNA (TR), whose secondary structure is essential for telomerase function and also contains the template that telomerase uses to add DNA. Previous studies in our lab have determined the secondary structure of TR in Tetrahymena thermophila (tTR) in solution and bound to the telomerase reverse transcriptase (TERT) using a single nucleotide resolution technique called selective 2’hydroxyl acylation analyzed by primer extension (SHAPE). Three uridine residues in Stem IV of tTR, which is important for nucleotide addition, exhibit similar flexibilities but only one U bulges out. We designed tTR mutants with C-G base pairs that would make each individual U bulge in order to see the effect of specific bulged U’s on telomerase activity and RNA structure. Telomerase assays showed decreased activity in the mutants compared to wildtype, while SHAPE analysis revealed changes in local secondary structure that may contribute to improper global RNA folding and telomerase function.

 

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