The ribonucleoprotein (RNP) enzyme telomerase is required for replication of eukaryotic chromosomal termini. The RNA moiety of telomerase is essential for enzyme function and provides the template for telomeric DNA synthesis. However, the roles of its nontemplate domains have not been explored. Here we demonstrate that a novel interspecies telomerase RNA swap in vivo creates a functional but aberrant telomerase. Telomerase RNA from the ciliate Glaucoma chattoni was expressed in Tetrahymena thermophila cells. The telomerase RNAs from these two species have almost superimposable secondary structures. The template region base sequence is identical in the two RNAs, but elsewhere their sequences differ by 49%. This hybrid telomerase RNP was enzymatically active but added only short stretches of telomeric repeat tracts in vivo and in vitro. This new enzyme also had a strong, aberrant DNA cleavage activity in vitro. Thus, molecular interactions in the RNP involving nontemplate RNA domains affect specific aspects of telomerase enzyme function, raising the possibility that they may regulate telomerase activity.The G-rich telomeric DNA tracts found at most eukaryotic chromosomal termini are added by a specialized component of the cellular DNA replication machinery, the ribonucleoprotein (RNP) reverse transcriptase telomerase. Telomeric DNA has numerous important functions and properties, including allowing completion of DNA replication, mediating chromosome stability (1), and affecting mitotic chromosome separation (2). Therefore, telomere synthesis and maintenance are crucial aspects of stable genomic inheritance in eukaryotes. The RNA moiety of the telomerase RNP is essential for enzymatic function and contains an internal sequence that templates telomeric DNA polymerization (3). Studies of template point mutations also have revealed other critical roles for specific template residues in enzyme action (4, 5). However, little is known about the function of other portions of telomerase RNA.Telomerase RNAs in ciliated protozoa (3, 6, 7), yeasts (8, 9), and mammals (10, 11) have diverged rapidly in primary sequence. Phylogenetic (12-14) and RNA conformational analyses (15, 16) of the small (Ϸ150-200 nt) ciliate telomerase RNAs have shown that their secondary structures are highly conserved. Comparison of 13 telomerase RNAs from one group of ciliates has shown a strikingly bipartite sequence arrangement; they all contain a 17-nt stretch of identical sequence centered on the template domain, and the primary sequence of the rest of the RNA differs by up to 76% yet preserves similar structural domains (12,14). To investigate directly how this nontemplate portion of telomerase RNA affects enzymatic function, we tested whether the Ϸ160-nt telomerase RNAs in the ciliates Tetrahymena thermophila and Glaucoma chattoni were functionally interchangeable in vivo. These two telomerase RNAs share an identical 23-base sequence in and around the template domain. In contrast, the rest of the RNA sequence, which has an almost identic...