Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for viable mutants

Telomerase is a ribonucleoprotein complex that adds telomeric DNA repeats t o the ends of most eukaryotic chromosomes. The reverse transcriptase subuni t of telomerase (TERT) differs from retroviral reverse transcriptases in ha ving a long basic amino-terminal extension. We made a large library contain ing random mutations in the amino terminus of the EST2 gene, which encodes the Saccharomyces cerevisiae TERT, and selected functional alleles by their ability to rescue senescence of telomerase-negative cells. Through analysi s of 265 mutations, the amino terminus of Est2p was found to contain at lea st four essential regions. This domain structure was verified by a combinat ion of deletion and alanine-block mutations. Mutations within two essential domains of the protein reduced RNA binding, suggesting that the amino term inus of Est2p makes important contacts with the intrinsic RNA component of telomerase. A mutant close to the amino terminus retained RNA binding and i n vitro enzymatic activity but was defective in vivo, suggesting a role in interaction with Other macromolecular components of telomerase.