The telomerase ribonucleoprotein (RNP) is required for maintaining telomeres, the specialized nucleoprotein structures that protect eukaryotic chromosome ends from aberrant processing and deleterious end-to-end fusion events. Telomerase catalyzes processive extension of telomere DNA using a unique catalytic mechanism that requires a strong functional interdependence of the telomerase RNA, telomerase reverse transcriptase, and several additional protein subunits. The primary objective of this proposal is to elucidate how conserved structural RNA and protein domains coordinate the processes of telomerase RNP assembly, catalysis, and regulation. To address the substantial challenges associated with structural analysis of telomerase we will utilize a multi-faceted experimental strategy that combines single-molecule biophysical techniques paired with computational, biochemical, and high-resolution structural approaches. We will study human telomerase and the enzyme from the model system Tetrahymena thermophila.
In aim 1, we will employ x-ray crystallography, single-molecule Frster resonance energy transfer (smFRET), and biochemical probing to characterize local and global structural features of the telomerase complex. Distance constraints furnished by these experiments will aid telomerase structural modeling in collaboration with Nikolai Ulyanov (UCSF).
In aim 2, we will use smFRET and small angle x-ray scattering (SAXS) to analyze the role(s) of telomere DNA structure and telomerase-associated proteins (POT1-TPP1, human; Teb1, Tetrahymena) in the regulation of telomerase-DNA interactions and catalysis. This work will be conducted in collaboration with Kathleen Collins (UCB) and Greg Hura (LBNL).
In aim 3, we will exploit several novel single-molecule assays to critically evaluate existing models for telomerase conformational dynamics during processive telomere DNA synthesis.

Public Health Relevance

Telomerase is an essential enzyme required for maintaining the protective capping structures found at chromosome ends called telomeres. Telomerase defects arise in the majority (~90%) of human cancers and several premature aging syndromes. Thus, the goal of developing novel approaches for diagnosing and treating telomerase-associated diseases motivates efforts to better understand the mechanism and regulation of telomerase. To this end, this proposal aims to illuminate the structural properties of telomerase that underlie its unique cellular activity.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Ainsztein, Alexandra M
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University of California Santa Cruz
Schools of Arts and Sciences
Santa Cruz
United States
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Shastry, Shankar; Steinberg-Neifach, Olga; Lue, Neal et al. (2018) Direct observation of nucleic acid binding dynamics by the telomerase essential N-terminal domain. Nucleic Acids Res 46:3088-3102
Musgrove, Cherie; Jansson, Linnea I; Stone, Michael D (2018) New perspectives on telomerase RNA structure and function. Wiley Interdiscip Rev RNA 9:
Parks, Joseph W; Stone, Michael D (2017) Single-Molecule Studies of Telomeres and Telomerase. Annu Rev Biophys 46:357-377
Parks, Joseph W; Kappel, Kalli; Das, Rhiju et al. (2017) Single-molecule FRET-Rosetta reveals RNA structural rearrangements during human telomerase catalysis. RNA 23:175-188
Long, Xi; Parks, Joseph W; Stone, Michael D (2016) Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid. Methods 105:16-25
Akiyama, Benjamin M; Parks, Joseph W; Stone, Michael D (2015) The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA-DNA hybrids. Nucleic Acids Res 43:5537-49
Jansson, Linnea I; Akiyama, Ben M; Ooms, Alexandra et al. (2015) Structural basis of template-boundary definition in Tetrahymena telomerase. Nat Struct Mol Biol 22:883-8
Parks, Joseph W; Stone, Michael D (2014) Coordinated DNA dynamics during the human telomerase catalytic cycle. Nat Commun 5:4146
Long, Xi; Stone, Michael D (2013) Kinetic partitioning modulates human telomere DNA G-quadruplex structural polymorphism. PLoS One 8:e83420
Akiyama, Benjamin M; Gomez, Anastassia; Stone, Michael D (2013) A conserved motif in Tetrahymena thermophila telomerase reverse transcriptase is proximal to the RNA template and is essential for boundary definition. J Biol Chem 288:22141-9

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