Telomerase RNA function in recombinant Tetrahymena telomerase

Telomerase is a ribonucleoprotein reverse transcriptase specialized for use of a sequence within its integral RNA component as the template for DNA sy nthesis. Telomerase adds telomeric simple sequence repeats to single-strand ed primers in vitro or chromosome ends in vivo. We have investigated the se quences and structures of recombinant Tetrahymena thermophila telomerase RN A necessary for physical association and activity with the catalytic protei n subunit expressed in rabbit reticulocyte lysate. In contrast with previou s results using another reconstitution method, we find that phylogeneticall y conserved primary sequences and a phylogenetically nonconserved secondary structure are essential for telomerase RNA function. Telomerase RNA bindin g to the catalytic protein subunit requires sequences 5' of the template an d is highly sequence specific. Other telomerase RNA sequences are required for enzyme activity and proper template use but not for protein interaction affinity. In addition, we demonstrate that the production of active recomb inant telomerase requires a factor in rabbit reticulocyte lysate that promo tes ribonucleoprotein assembly. These studies demonstrate multiple function s for the telomerase RNA and indicate that recombinant telomerase activity requires more than the catalytic protein and RNA components of the enzyme t hat have been identified to date.