This dissertation describes the structural characterization of the telomerase holoenzyme from the ciliate Tetrahymena thermophila using a combination of X-ray crystallography, cryogenic electron microscopy, and nuclear magnetic resonance spectroscopy. Telomerase is the ribonucleoprotein complex that iteratively reverse transcribes telomeric repeats to the ends of linear chromosomes in actively replicating eukaryotic cells. Previous biochemical and genetic studies of Tetrahymena telomerase holoenzyme identified the components TERT (the reverse transcriptase), TR (the internal RNA template), and six other accessory proteins (p65, Teb1, p50, p75, p45, and p19). Of these, p50, p75, p45, and p19 were largely uncharacterized and possessed no known homology to other proteins. A major focus of this work was to study p75-45-p19 which was known to form a trimeric complex. In the course of this work, crystal structures of p19 and the p45 C-terminal domain were solved and shown to be structurally homologous to the Ten1 and Stn1 proteins of the Replication Protein A (RPA)-like telomeric Ctc1-Stn1-Ten1 (CST) complex that is found in plants, vertebrates, and yeast, leading to the conclusion that p75-p45-p19 is Tetrahymena CST. In addition, the ongoing structural characterization of the p75 protein is discussed, in which a specific interaction between p75 and p50 is identified. The work in this thesis also contributed to the overall structural characterization of the entire holoenzyme through an electron microscopy (EM) collaboration with the Zhou lab where negative stain and cryogenic EM (cryo-EM) structures of Tetrahymena telomerase were solved to 25 and 9 Å resolution respectively. These EM structures led to the characterization and unambiguous placement of all of the then known subunits including the placement of a homology model of the p75-p45-p19 trimeric core, based on the structure of p19 and the structure of the RPA trimeric core, into the cryo-EM structure, further confirming the characterization of p75-p45-p19 as Tetrahymena CST. The cryo-EM structure also led to the identification of two new subunits Teb2 and Teb3 by mass spectrometry, which were determined to form another trimeric RPA-like subcomplex, TEB, with Teb1. The identification and characterization of these TEB proteins led to the discovery that Teb2 and Teb3 are shared subunits with Tetrahymena RPA and of a new RPA-like protein, Rlp2, which forms an alternative complex with Teb3.

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.

The S. cerevisiae RNase III enzyme Rnt1p preferentially binds to dsRNA hairpin substrates with a conserved (A/u)GNN tetraloop fold, via shape-specific interactions by its dsRBD helix α1 to the tetraloop minor groove. The solution structure of Rnt1p dsRBD bound to an AAGU-capped hairpin reveals that the tetraloop undergoes a structural rearrangement upon binding to Rnt1p dsRBD to adopt a backbone conformation that is essentially the same as the AGAA tetraloop, and indicates that a conserved recognition mode is used for all Rnt1p substrates. Comparison of free and RNA-bound Rnt1p dsRBD reveals that tetraloop-specific binding requires a conformational change in helix α1. To investigate whether conformational flexibility in the dsRBD regulates the binding specificity, I determined the backbone dynamics of the Rnt1p dsRBD in the free and AGAA hairpin-bound states using NMR spin relaxation experiments. The intrinsic μs-ms timescale dynamics of the dsRBD suggests that helix α1 undergoes conformational sampling in the free state, with large dynamics at some residues in the α1-β1 loop (α1- β1 hinge). These results, in combination with an RDC-refined solution structure of the free dsRBD, revealed that the Rnt1p dsRBD has an extended hydrophobic core comprising helix α1, the α1-β1 loop, and helix α3. Analysis of the backbone dynamics and structures of the free and bound dsRBD reveals that slow-timescale dynamics in the α1-β1 hinge are associated with concerted structural changes in the extended hydrophobic core that govern binding of helix α1 to AGAA tetraloops. The dynamic behavior of the dsRBD bound to a longer AGAA hairpin reveals that dynamics within the hydrophobic core differentiate between specific and non-specific sites. Mutations of residues in the α1-β1 hinge result in changes to the dsRBD stability and RNA-binding affinity, and cause defects in snoRNA processing in vivo. These results reveal that dynamics in the extended hydrophobic core are important for binding site selection by the Rnt1p dsRBD.