Telomeres are the specialized DNA-protein structures that cap the ends of eukaryotic chromosomes and serve an essential role in maintaining chromosome stability and proper nuclear architecture. Highly conserved throughout evolution, telomeric DNA consists of simple tandemly repeated sequences. A novel ribonucleoprotein (RNP) enzyme called telomerase is responsible for synthesizing telomeric DNA. Within the telomerase RNA moiety is a sequence complementary to the telomeric repeats and this domain serves as the template for DNA synthesis. Thus, telomerase is a specialized type of reverse transcriptase. The long range goal of this project is to elucidate the mechanism of telomere synthesis by the telomerase enzyme. Currently, our understanding of this process is impeded by the lack of information concerning the components of the telomerase RNP. Although both RNA and protein are required for activity, the telomerase RNA has only been partially characterized, while the protein component(s) of this enzyme have not yet been identified. This application is aimed at further analysis of telomerase RNP structure by exploiting the component we have in hand, the RNA moiety. The experimental system chosen for these studies are hypotrichous ciliates, Euplotes and Oxytricha, two of the richest known sources of telomeres. There are two major goals in this proposal. The first is an examination of the telomerase RNA, analyzing the secondary structure of this molecule by phylogenetic and in vitro experimental approaches. In addition, we will also explore the function of the telomeric templating domain in the Oxytricha telomerase RNA. The second part of this proposal investigates RNA-protein interactions within the telomerase RNP. Two different approaches based on techniques developed for the analysis of other small nuclear RNP complexes will be used to identify proteins that are lightly associated with the telomerase RNA. The first will be to partially purify the Euplotes telomerase RNP using conventional column chromatography and equilibrium gradient centrifugation, following either enzymatic activity or the telomerase RNA component as a marker for the telomerase complex. Samples will be further purified by affinity selection of Euplotes telomerase RNP particles using oligonucleotides complementary to the telomerase RNA moiety. A second strategy to identify polypeptides associated with the telomerase RNA will assay for the assembly of proteins from partially purified telomerase preparations onto exogenous telomerase RNA transcripts in vitro. Proteins that bind to the telomerase RNA will be identified and further characterized.
