Telomerase is a specialized reverse transcriptase composed of an RNA subunit and at least one protein subunit. Our goal is to address the function and regulation of S. cerevisiae telomerase, focusing on the catalytic subunit, Est2. In addition to reverse transcriptase motifs, all Est2 homologs contain a basic domain and a novel telomerase-specific motif (T motif).
The first aim of this proposal is to determine the roles of the basic domain and the T motif by alanine scanning mutagenesis of these regions. The est2 mutants will be expressed in an est2-delta strain and analyzed for senescence and telomere length in vivo, and telomerase activity using an in vitro assay. The second, related aim of this proposal is to identify and characterize gene products that affect telomerase function in vivo, and specifically those gene products that physically interact with Est2. Taking advantage of a strain background that makes est mutations nearly synthetic lethal (V. Lundblad, unpublished results), conditional est2 mutants will be used to screen for extragenic suppressors, and the genes containing extragenic suppressors will be cloned and analyzed for their role in telomerase function. To determine if these suppressor gene products and current EST gene products (Estl, Est3, Cdc13) are telomerase-associated proteins, two complementary approaches will be taken. The first approach will employ gentle fractionation of extracts prepared from a yeast strain that expresses epitope-tagged versions of Est1, Est2, Est3 and Cdc13. Reciprocal immunoprecipitation and western analysis of the immune complexes will be used to identify gene products that fractionate with the telomerase RNP complex (Est2/Tlc1). In the second approach, Est2 will be expressed in reticulocyte lysates and candidate proteins will be tested for interaction with Est2 in vitro by reciprocal immunoprecipitation experiments. The potential involvement of the basic domain and T motif in mediating such interactions can be addressed by use of the alanine scanning est2 mutants in these experiments. These studies will provide important insight into the regulation of telomerase activity, and will test for the existence of a telomerase holoenzyme in yeast. The work initiated here also provides a detailed molecular analysis of the catalytic subunit of telomerase. This information may ultimately prove useful if telomerase-based therapeutics for human cancers are adopted.
