UCLA

This dissertation consists of four projects, three focused on Nuclear Magnetic Resonance (NMR) structural analysis applied to telomerase RNA (TER) and one on optimizing expression of telomerase proteins. Telomerase is the enzyme necessary for synthesis of telomere repeats at the 3' end of linear chromsomes. It has a minimal composition of TER and telomerase reverse transcriptase (TERT). TER includes the template used for copying the telomeric repeat and an adjacent pseudoknot which is important for telomerase function. The structure of the human TER pseudoknot revealed it contains a triple helix with tertiary interactions that are essential for telomerase activity. The goal of the first project was to further study pseudoknot structure to gain insights into conserved features and pseudoknot function in telomerase. To this end, the NMR solution structure of the yeast K. lactis TER pseudoknot was determined. This pseudoknot contains an extended pyrimidine motif triple helix with a C-G-C+ triple and three bulge nucleotides to maintain continuous base pairing and stacking interactions through the stems. Despite differences in sequence and base triples, the human and yeast pseudoknots have a remarkably similar tertiary shape, indicating a conserved function. In the second project, the NMR solution structure of the ciliate Tetrahymena thermophila TER pseudoknot was determined. The Tetrahymena pseudoknot (tetPK) is more compact than the pseudoknots of human and yeast, however it maintains the conserved features of stacked stems and base triple interactions. TetPK contains a unique A-G-C base triple, which is shown to be important for pseudoknot stability and telomerase activity. The folding of tetPK was studied in the context of full length TER, where its formation was shown to be disrupted by competition with alternate structures. The third project analyzed NMR structure calculations of A-form RNA helices. Two parameters were determined to be essential for getting accurate helical structures, distance restraints derived from sequential base-to-base NOEs and inclusion of accurate and sufficient residual dipolar couplings (RDCs). The fourth project involved protein expression of telomerase proteins for structure and functional study. For this, a new cloning system was designed termed diverse combination ligation indepenedent cloning (DC-LIC). DC-LIC combines the advantages of a number of gene cloning and protein expression techniques to simplify and optimize production of recombinant proteins from E. coli. DC-LIC was used to build a number of telomerase gene constructs with improved expression and solubility.