Project Summary/Abstract Telomeres, the physical ends of linear chromosomes, are composed of short repeating sequences (TTGGGG in the G-strand for Tetrahymena thermophila and TTAGGG in human) of double-strand DNA with a single-strand 3′ overhang of the G-strand and, in humans, six proteins collectively called shelterin. Of these, TPP1–POT1 associate with the 3′ overhang, where POT1 binds the G-strand and TPP1 functions to recruit telomerase via interaction with telomerase reverse transcriptase (TERT). The telomere DNA 3’-ends are replicated and maintained by telomerase, for the G-strand and subsequently DNA polymerase α–primase (PolαPrim) in complex with CTC1–STN1–TEN1 (CST), for the C-strand. In Tetrahymena, orthologous proteins p50 (TPP1), Teb1 (POT1), and CST are constitutively associated with telomerase. Mutations that affect their activity or regulation have profound effects on human health. Recent cryoEM structures and other studies on Tetrahymena and human telomerase and these interacting partners set the stage for our goals to illuminate the detailed mechanism and regulation of G-strand and C-strand synthesis at telomeres. Our focus will be on two broad aspects of human and Tetrahymena telomerase and telomere structural biology: (A) telomerase mechanism, assembly, and disease and (B) C-strand synthesis by CST–PolαPrim. Our research approach will primarily involve complementary applications of cryoEM and NMR spectroscopy along with rigorous biochemistry and molecular biology. Results on the two organisms will provide complementary information on shared structures and mechanisms while also providing an evolutionary perspective for divergent organisms. Additionally, (C) we will extend from single particle cryoEM to cryoET to visualize telomerase at telomeres in situ.