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 to the ends of most eukaryotic chromosomes. The reverse transcriptase subunit of telomerase (TERT) differs from retroviral reverse transcriptases in having a long basic amino-terminal extension. We made a large library containing 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 analysis of 265 mutations, the amino terminus of Est2p was found to contain at least four essential regions. This domain structure was verified by a combination of deletion and alanine-block mutations. Mutations within two essential domains of the protein reduced RNA binding, suggesting that the amino terminus of Est2p makes important contacts with the intrinsic RNA component of telomerase. A mutant close to the amino terminus retained RNA binding and in vitro enzymatic activity but was defective in vivo, suggesting a role in interaction with other macromolecular components of telomerase.
Friedman
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KL
Department of Chemistry and Biochemistry and Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309-0215, USA.
Cech
T R
TR
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D012313:RNA; D012331:RNA, Fungal / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D019098:Telomerase / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D004268:DNA-Binding Proteins; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C422952:telomerase RNA; D012313:RNA; C509185:EST2 protein, S cerevisiae; D019098:Telomerase; D000409:Alanine