A Hybrid Methods Approach to Determine the Structure of Tetrahymena Telomerase

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Telomerase is an RNA-protein complex that extends the ends of linear chromosomes, and is a highly regulated determinant of cellular aging, stem cell renewal, and tumorigenesis. We are using a combination of NMR spectroscopy, X-ray crystallography, mass spectrometry, and electron microscopy to study the structure and function of telomerase. We previously reported the 3D structure of endogenously assembled Tetrahymena thermophila telomerase holoenzyme at 25 A resolution using negative stain electron microscopy (EM) (Jiang et al Nature 2013). Six of the 7 protein subunits and the stem-loop 2 region of telomerase RNA (TER) were localized in the 3D structure by affinity labeling. Among the holoenzyme proteins, p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution correlates activity with structure. This first physical and functional network architecture of a telomerase holoenzyme provided the first view into the structure of the telomerase reverse transcriptase (TERT)-TER-p65 catalytic core and revealed the organization of holoenzyme subunits that confer processivity and bridge telomerase to telomeres. More recently, we determined the cryoelectron microscopy structure of Tetrahymena telomerase at ∼9-A resolution. In addition to the 7 known holoenzyme proteins, 2 new proteins are identified, which form a complex (TEB) with single-stranded telomere DNA-binding protein Teb1, paralogous to heterotrimeric Replication Protein A (RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST. This study reveals the paths of TER in the TERT-TER-p65 catalytic core and ssDNA exit, extensive subunit interactions of TERT essential N-terminal domain, p50, and TEB, and new subunit identities and structures, including p19 and p45C crystal structures, providing unprecedented structural and mechanistic insights into telomerase holoenzyme function.