InTERTpreting telomerase structure and function

Abstract

The Nobel Prize in Physiology or Medicine was recently awarded to Elizabeth Blackburn, Carol Greider and Jack Szostak for their pioneering studies on chromosome termini (telomeres) and their discovery of telomerase, the enzyme that synthesizes telomeres. Telomerase is a unique cellular reverse transcriptase that contains an integral RNA subunit, the telomerase RNA and a catalytic protein subunit, the telomerase reverse transcriptase (TERT), as well as several species-specific accessory proteins. Telomerase is essential for genome stability and is associated with a broad spectrum of human diseases including various forms of cancer, bone marrow failure and pulmonary fibrosis. A better understanding of telomerase structure and function will shed important insights into how this enzyme contributes to human disease. To this end, a series of high-resolution structural studies have provided critical information on TERT architecture and may ultimately elucidate novel targets for therapeutic intervention. In this review, we discuss the current knowledge of TERT structure and function, revealed through the detailed analysis of TERT from model organisms. To emphasize the physiological importance of telomeres and telomerase, we also present a general discussion of the human diseases associated with telomerase dysfunction.

Figure 1.

Structural organization of the TERT. (A) Predicted linear architecture of hTERT. In most organisms, TERT contains a long N-terminal extension (NTE), a central catalytic RT domain and a short C-terminal extension (CTE). Green boxes indicate the predicted locations of the telomerase essential N-terminal (TEN) domain and the telomerase-specific motifs CP, QFP and TS. Blue boxes indicate the TRBD, containing the CP motif, QFP motif and part of the TS motif. An unstructured linker region connects the TEN domain and TRBD. Orange and tan boxes represent the seven evolutionarily conserved motifs in the RT domain (1, 2, A, B′, C, D, E) and a red box illustrates the telomerase-specific IFD. The CTE contains four blocks of conserved amino acids, which are shown as pink boxes (E-I, E-II, E-III, E-IV). (B) Domain organization of the protein predicted to be T. castaneum TERT (cartoon and surface representation), reprinted with permission from ref. (42). The protein is organized into a ring-shaped structure, containing hallmark ‘thumb’ (red), ‘palm’ (tan) and ‘fingers’ (orange) motifs. The telomerase-specific TRBD is shown in violet. The color scheme used in (A) corresponds to that shown in (B).

Figure 2.

Molecular models of T. thermophila (A) and T. castaneum (B–D) TERT. (A) Surface representation of the isolated TRBD from T. thermophila TERT, reprinted with permission from ref. (56). This cartoon shows the two asymmetric lobes that comprise the TRBD. Amino acids that form the TS motif are shown in pink and those that comprise the CP motif are indicated in blue. (B) Model of the protein suggested to be T. castaneum TERT (surface representation) in complex with the telomerase RNA subunit (dark green) and single-stranded telomeric DNA (dark purple), reprinted with permission from ref. (42). The hallmark motifs of the RT domain are illustrated as follows: motif 1 (red), 2 (grey), A (green), B′ (dark purple), C (blue), D (dark blue), E (magenta) and IFD (light blue). The TRBD comprises residues from the CP (yellow) and TS (cyan) motifs. Structural elements of the TERT C-terminal extension (light green) are predicted to stabilize and orientate the TERT–RNA–DNA complex. (C) Domain folds of the predicted T. castaneum TERT RT domain, colored as in (B) and reprinted with permission from ref. (42). Elements that form the palm domain are colored in tan and those that form the fingers domain are shown in orange. (D) The active site and nucleotide-binding pocket of T. castaneum TERT, reprinted with permission from ref. (42). This figure shows the predicted location of three invariant aspartic acids (D251, D343 and D344) that form the catalytic triad of the active site in complex with a modeled nucleotide of ATP (black stick). The RT motifs are colored as in (B).

Figure 3.

Schematic diagram of the telomerase reaction cycle. This figure summarizes the fundamental steps of the telomerase reaction cycle: initial primer recognition and binding, nucleotide addition and translocation. The telomerase RNA (TR) subunit is represented by a black line (not drawn to scale). The TR supplies the template (grey rectangle) for telomere synthesis. DNA synthesis is catalyzed by the TERT. TERT contains a TEN domain (pink sphere), a TRBD (purple oval), RT domain (blue sphere) and CTE (green oval). The TEN domain contains a unique ssDNA-binding region, called the telomerase anchor site (transparent rectangle). The red line represents telomeric ssDNA. Telomerase binds the telomeric ssDNA such that the 3′-end is aligned with the TR template in the active site (white star) and the 5′-end is positioned within the telomerase anchor site. Telomerase reverse transcribes the template region, 1 nt at a time (nucleotide addition), until reaching the 5′-template boundary element (top right). At this point, a translocation step repositions the new DNA 3′-end within the template for a second round of telomere synthesis (bottom). Conformational changes within the telomerase holoenzyme are believed to facilitate nucleotide and repeat addition processivity.