Telomerase is a ribonucleoprotein complex responsible for extending the dG-rich strand of the telomere terminal repeats. It employs a small segment of a stably associated RNA component as template, and pre-existing chromosomal ends as primers to accomplish reverse transcription. Telomerase activity is specifically activated in cancer cells, and promotes neoplastic transformation by conferring cells with unlimited replicative potentials. Specific inactivation of telomerase in tumor cells leads to telomere shortening and apoptosis, validating the potential of telomerase inhibitors in anti-cancer therapy. The long-term objective of this research plan is to establish a detailed molecular understanding of telomerase structure, mechanism, assembly and regulation. This proposal focuses on a key component of the telomerase complex, the reverse transcriptase polypeptide (known as TERT). Located within the C-terminal half of this protein are essential reverse-transcriptase-like motifs that participate in polymerization. Comparative sequence analysis by others and the principal investigator revealed in the N-terminal half of the protein four novel conserved motifs (named GQ, CP, QFP, and T based primarily on key invariant residues within each motif) that are likely to be critical to its function. The principal investigator hypothesizes that these motifs mediate crucial activities of TERT, such as RNA-binding, protein stability, complex assembly, interaction with regulatory factors, and reverse transcription. The goals of this proposal are directed toward elucidating the precise molecular functions of all four motifs. The methodology involves biochemical and genetic analysis of telomerase complex bearing deletion and substitution mutations, as well as biophysical and structural characterization of critical protein domains in isolation. Given the wealth of available biochemical and genetic tools, the budding yeast Saccharomyces cerevisiae represents an ideal model system for undertaking such an endeavor. Furthermore, because of the conserved nature of the motifs being analyzed, the findings should provide general insights on telomerase structure and mechanisms.
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