塩見研公開ラボセミナー
A hybrid methods approach to determine the structure of Tetrahymena telomerase
Juli Feigon（UCLA, Los Angeles, CA USA）
2015年11月09日(月) 17：00-18：00 理学部3号館 327号室
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 Å 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-Å resolution (Jiang et al Science 2015). 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.
