title: Reconstitution of human telomerase activity in vitro.
abstract: Telomerase is a ribonucleoprotein enzyme complex that adds single-stranded telomere DNA to chromosome ends [1]. The RNA component of telomerase contains the template for telomeric DNA addition and is essential for activity [1,2]. Telomerase proteins have been identified in ciliates, yeast and mammals [3-12]. In Saccharomyces cerevisiae, the Est2 protein is homologous to the 123 kDa reverse transcriptase subunit of Euplotes telomerase, and is essential for telomerase activity [8]. In humans, telomerase activity is associated with the telomerase RNA hTR [13], the telomerase RNA-binding protein TP1/TLP1 [5,12] and the TP2 protein encoded by the human EST2 homolog [12] (also known as TRT1, hEST2 or TCS1 [9-11]). The minimal complex sufficient for activity is, however, unknown. We have reconstituted human telomerase activity in reticulocyte lysates and find that only exogenous hTR and TP2 are required for telomerase activity in vitro. Recognition of telomerase RNA by TP2 was species specific, and nucleotides 10-159 of hTR were sufficient for telomerase activity. Telomerase activity immunoprecipitated from the reticulocyte lysate contained hTR and recombinant TP2. Substitution of conserved amino acid residues in the reverse transcriptase domain of TP2 completely abolished telomerase activity. We suggest that TP2 and hTR might represent the minimal catalytic core of human telomerase.
authors.affiliation: Ontario Cancer Institute-Amgen Institute, Department of Medical Biophysics, University of Toronto, 620 University Avenue, Toronto, Ontario, M5G 2C1, Canada.
authors.forename: T L
authors.initials: TL
authors.lastname: Beattie
authors.forename: W
authors.initials: W
authors.lastname: Zhou
authors.forename: M O
authors.initials: MO
authors.lastname: Robinson
authors.forename: L
authors.initials: L
authors.lastname: Harrington
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D002384:Catalysis; D002474:Cell-Free System; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D011506:Proteins / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012156:Reticulocytes; D029701:Saccharomyces cerevisiae Proteins; D019098:Telomerase / Q000737:chemistry / Q000378:metabolism*; D013698:Templates, Genetic
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011506:Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C104342:TEP1 protein, human; C422952:telomerase RNA; D012313:RNA; C509185:EST2 protein, S cerevisiae; C509186:TERT protein, human; D019098:Telomerase