Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3′ ends of most eukaryotic chromosomes¹. Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression^2,3. Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA–protein and RNA–protein interactions and of the mechanisms and recruitment involved remain limited^4,5,6. Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5′ and 3′ template boundaries, handling of the template–DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP1^7,8) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5′ and 3′ ends and the conserved pseudoknot to facilitate assembly of the TERT–TER catalytic core.

端粒酶在逆转录酶中是独一无二的，因为它含有一个非编码 RNA（称为端粒酶 RNA (TER)），它包括一个短模板，用于在大多数真核生物染色体¹的 3' 末端进行富含 G 的端粒 DNA 重复序列的持续合成. 端粒酶维持基因组完整性，其活性或失调是人类寿命、干细胞更新和癌症进展的关键决定因素^2,3。以前的低温电子显微镜结构已经确定了四膜虫和人类端粒酶的一般结构、蛋白质成分和化学计量，但我们对 DNA-蛋白质和 RNA-蛋白质相互作用的细节以及所涉及的机制和募集的理解仍然有限^{4,5, 6}. 在这里，我们报告了在核苷酸添加的不同步骤中具有端粒 DNA的活性四膜虫端粒酶的低温电子显微镜结构。端粒酶逆转录酶 (TERT)、TER 和 DNA 之间的相互作用揭示了确定 5' 和 3' 模板边界、处理模板-DNA 双链体和在核苷酸添加过程中分离产物链的结构基础。TERT 与端粒酶蛋白 p50（人类 TPP1 ^{7,8的同源物）之间的结构和结合界面}) 定义端粒酶激活和招募到端粒所需的保守相互作用。端粒酶 La 相关蛋白 p65 重塑了 TER 的几个区域，桥接 5' 和 3' 末端以及保守的假结以促进 TERT-TER 催化核心的组装。