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Structural and Functional Characterization of the Tetrahymena Telomerase Holoenzyme

Abstract

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.

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