A common hallmark of human cancers is the overexpression of telomerase, a ribonucleoprotein complex that is responsible for maintaining the length and integrity of chromosome ends. Telomere length deregulation and telomerase activation is an early, and perhaps necessary, step in cancer cell evolution. Here we present the high-resolution structure of the Tribolium castaneum catalytic subunit of telomerase, TERT. The protein consists of three highly conserved domains, organized into a ring-like structure that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven to eight bases of double-stranded nucleic acid. Modelling of an RNA–DNA heteroduplex in the interior of this ring demonstrates a perfect fit between the protein and the nucleic acid substrate, and positions the 3′-end of the DNA primer at the active site of the enzyme, providing evidence for the formation of an active telomerase elongation complex.

•Structure of a vertebrate telomerase TRBD•Identification of a conserved motif (TFLY) in vertebrate telomerase N-terminal linker•The TFLY motif contributes to the T-CP pocket formation and TBE binding•T and TFLY motif TBE binding required for telomerase activity and processivityTelomerase is a ribonucleoprotein reverse transcriptase that replicates the ends of chromosomes, thus maintaining genome stability. Telomerase ribonucleoprotein assembly is primarily mediated by the RNA binding domain (TRBD) of the enzyme. Here we present the high-resolution TRBD structure of the vertebrate, Takifugu rubripes (trTRBD). The structure shows that with the exception of the N-terminal linker, the trTRBD is conserved with the Tribolium castaneum and Tetrahymena thermophila TRBDs, suggesting evolutionary conservation across species. The structure provides a view of the structural organization of the vertebrate-specific VSR motif that binds the activation domain (CR4/5) of the RNA component of telomerase. It also reveals a motif (TFLY) that forms part of the T-CP pocket implicated in template boundary element (TBE) binding. Mutant proteins of conserved residues that consist of part of the T and TFLY motifs disrupt trTRBD-TBE binding and telomerase activity and processivity, supporting an essential role of these motifs in telomerase RNP assembly and function.

•Cancer therapeutics targeting telomerase•Structure of TERT in complex with the allosteric inhibitor BIBR1532•BIBR1532 binds the thumb domain of TERT•BIBR1532 inhibits telomerase function by disrupting TERT-RNA bindingBIBR1532 is a highly specific telomerase inhibitor, although the molecular basis for inhibition is unknown. Here we present the crystal structure of BIBR1532 bound to Tribolium castaneum catalytic subunit of telomerase (tcTERT). BIBR1532 binds to a conserved hydrophobic pocket (FVYL motif) on the outer surface of the thumb domain. The FVYL motif is near TRBD residues that bind the activation domain (CR4/5) of hTER. RNA binding assays show that the human TERT (hTERT) thumb domain binds the P6.1 stem loop of CR4/5 in vitro. hTERT mutations of the FVYL pocket alter wild-type CR4/5 binding and cause telomere attrition in cells. Furthermore, the hTERT FVYL mutations V1025F, N1028H, and V1090M are implicated in dyskeratosis congenita and aplastic anemia, further supporting the biological and clinical relevance of this novel motif. We propose that CR4/5 contacts with the telomerase thumb domain contribute to telomerase ribonucleoprotein assembly and promote enzymatic activity.

Telomerase, a ribonucleoprotein complex, replicates the linear ends of eukaryotic chromosomes, thus taking care of the “end of replication problem.” TERT contains an essential and universally conserved domain (TRBD) that makes extensive contacts with the RNA (TER) component of the holoenzyme, and this interaction is thought to facilitate TERT/TER assembly and repeat-addition processivity. Here, we present a high-resolution structure of TRBD from Tetrahymena thermophila. The nearly all-helical structure comprises a nucleic acid-binding fold suitable for TER binding. An extended pocket on the surface of the protein, formed by two conserved motifs (CP and T motifs) comprises TRBD's RNA-binding pocket. The width and the chemical nature of this pocket suggest that it binds both single- and double-stranded RNA, possibly stem I, and the template boundary element (TBE). Moreover, the structure provides clues into the role of this domain in TERT/TER stabilization and telomerase repeat-addition processivity.