Beattie T L, Zhou W, Robinson M O, Harrington L
Ontario Cancer Institute/Amgen Institute, Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2C1 Canada.
Mol Biol Cell. 2000 Oct;11(10):3329-40. doi: 10.1091/mbc.11.10.3329.
The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs.
人类端粒酶的最小活性核心被推测包含两个组分，即端粒酶RNA hTER和端粒酶逆转录酶hTERT。体外重建人类端粒酶活性有助于鉴定端粒酶RNA中的序列以及hTERT的逆转录基序，这些对于端粒酶活性至关重要。然而，hTERT逆转录结构域之外的残基的确切作用尚不清楚。在此，我们已经描绘出hTERT内几个对于端粒酶催化、引物使用以及与端粒酶RNA和端粒酶相关蛋白TEP1相互作用很重要的区域。特别地，hTERT氨基和羧基末端的某些缺失，尽管保留了与端粒酶RNA和TEP1的相互作用，但在体外和体内均完全无活性。此外，缺乏氨基末端但能够结合端粒酶RNA的hTERT截短体在延伸端粒和非端粒引物的能力方面严重受损。这些结果表明，端粒酶RNA与hTERT的相互作用在功能上可能与聚合反应解偶联，并且hTERT逆转录结构域之外存在对端粒酶活性和引物使用至关重要的区域。这些结果证实，人类端粒酶逆转录酶具有独特的聚合决定因素，使其有别于其他逆转录酶。
