Research Article

Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions

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Science  30 Oct 2015:
Vol. 350, Issue 6260, aab4070
DOI: 10.1126/science.aab4070

Chromosome-capping enzyme complex

Telomeres cap and protect the ends of our chromosomes. The telomerase complex helps maintain the telomere DNA repeat sequences. Telomerase consists of an RNA and a number of protein subunits. Jiang et al. used cryo–electron microscopy and x-ray crystallography to determine the structure of the Tetrahymena telomerase complex. The telomerase is made up of three subcomplexes, which include two previously unknown protein subunits in addition to the seven known subunits. The structures also reveal the path of the RNA component in the telomerase catalytic core.

Science, this issue p. 10.1126/science.aab4070

Structured Abstract


Telomerase is a ribonucleoprotein complex that extends the telomere DNA at the 3′ ends of linear chromosomes, thereby counteracting the loss of DNA from replication and nucleolytic processing. Although telomerase is largely inactive in somatic cells, it is active in stem cells and highly active in most cancer cell lines, where its activity is necessary for the cells’ immortal phenotype. Thus, telomerase is an important regulator of aging, tumorigenesis, and stem cell renewal. Telomerase uses a template contained within the integral telomerase RNA (TER) and a telomerase reverse transcriptase (TERT) to synthesize multiple copies of the G-strand telomere repeat, which is TTGGGG in Tetrahymena. Telomerase can synthesize telomere repeat DNA in vitro with only TERT and TER, but physiological function requires a variety of other proteins. Telomerase recruitment to telomeres is regulated by the cell cycle, where its activity requires interplay between telomere end-protection and telomerase proteins. Telomere end-maintenance also requires coordinated recruitment of telomerase and DNA polymerase α for synthesis of G and C strands, respectively.


A detailed mechanistic description of telomerase and its interaction at telomeres has been hampered by a lack of structural models, due to difficulties in obtaining samples of sufficient quantity and quality, as well as subunit complexity and flexibility and low sequence identity among subunits from different organisms. Unlike yeast and mammalian telomerase, Tetrahymena telomerase is constitutively assembled, making it possible to purify from cells and study all holoenzyme components in a stable complex. In addition to TERT and TER, the Tetrahymena telomerase holoenzyme contains the known proteins p65, p75, p45, p19, p50, and Teb1, a telomeric G-strand binding protein that is paralogous to the large subunit of heterotrimeric replication protein A (RPA). TER contains a template/pseudoknot domain enclosing a template with sequence complementarity to ~1.5 telomere repeats and a separate activating domain. Though TER is essential for activity, the physical arrangement of TER on TERT has remained largely unknown, as have details of protein subunit interactions.


We report cryo–electron microscopy (cryo-EM) structures at ~9 Å resolution and pseudoatomic modeling of Tetrahymena telomerase, crystal structures of p19 and p45C, and a nuclear magnetic resonance structure of the TER pseudoknot. Two newly identified and functionally distinct RPA-related complexes, Teb1-Teb2-Teb3 (TEB) and p75-p45-p19, are connected to the TERT-TER-p65 catalytic core by p50. The presence of Teb2 and Teb3 is confirmed by mass spectrometry. p19 and p45 are structurally homologous to Ten1 and Stn1, respectively, which are found in the telomere end-binding complex CST (CTC1-STN1-TEN1) that recruits DNA polymerase α. The path of TER on TERT and the location of the TERT essential N-terminal domain (TEN) are revealed, providing mechanistic insights into telomerase function. A network of interactions between TEN, p50, and TEB regulates and enhances activity. p50-Teb1 appears to be structurally and functionally homologous to human TPP1-POT1. Negative-stain electron microscopy of labeled telomeric DNA bound to telomerase reveals the exit path from the template.


The structure of the Tetrahymena telomerase catalytic core and our identification of telomerase holoenzyme subcomplexes that are homologous to those found at mammalian, plant, and yeast telomeres provide new mechanistic insights and suggest commonalities of telomerase interaction, action, and regulation at telomeres.

Two views of the Tetrahymena telomerase holoenzyme.

The top left image shows a front view of pseudoatomic models as spacefill superimposed on the cryo-EM map. Models of protein domains connected by flexible linkers and not visible in the cryo-EM map are illustrated as ribbons. The bottom right image shows a back-view schematic with the telomerase-bound telomeric G strand connected to a chromosome via double-stranded telomere DNA. Teb1AB domains are presumed to be ordered when DNA bound.


Telomerase helps maintain telomeres by processive synthesis of telomere repeat DNA at their 3′-ends, using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). We report the cryo–electron microscopy structure of Tetrahymena telomerase at ~9 angstrom resolution. In addition to seven known holoenzyme proteins, we identify two additional proteins that 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 (CTC1-STN1-TEN1). This study reveals the paths of TER in the TERT-TER-p65 catalytic core and single-stranded DNA exit; extensive subunit interactions of the TERT essential N-terminal domain, p50, and TEB; and other subunit identities and structures, including p19 and p45C crystal structures. Our findings provide structural and mechanistic insights into telomerase holoenzyme function.

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