Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA
Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA
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DOI：
10.1002/j.1460-2075.1996.tb00979.x
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发表时间：
1996-11-01
期刊：
影响因子：
11.4
通讯作者：
Greider, CW
中科院分区：
文献类型：
--
作者：
Autexier, C;Pruzan, R;Greider, CW
端粒酶是一种核糖核蛋白，它通过添加TTAGGG重复序列来催化端粒的延长，端粒酶的激活与人类细胞的永生化和癌症有关，为了研究端粒酶的作用机制，我们建立了一种功能性的体外重建实验，在通过微球菌核酸酶消化部分纯化的人端粒酶去除必需的445个核苷酸的人端粒酶RNA（hTR）后，加入体外转录的hTR重建端粒酶活性。该活性依赖于hTR并对hTR具有特异性。通过对hTR 5'端和3'端的截短，鉴定出一个与纤毛虫端粒酶RNA全长相似的功能区，该功能区位于1-203位之间。此外，我们还发现模板区5'端的1-44位残基，（残基46-56）对于活性不是必需的，表明最小功能区位于残基44-203之间，180-189或190-199的序列几乎完全消除了hTR在端粒酶活性重建中发挥作用的能力，表明这30个核苷酸区域内的序列或结构是活性所需的，可能是通过结合端粒酶蛋白组分。
Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans, Activation of telomerase is often associated with immortalization of human cells and cancer, To dissect the human telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay, After removal of the essential 445 nucleotide human telomerase RNA (hTR) by micrococcal nuclease digestion of partially purified human telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity. The activity was dependent upon and specific to hTR. Using this assay, truncations at the 5' and 3' ends of hTR identified a functional region of hTR, similar in size to the full-length telomerase RNAs from ciliates, This region is located between positions 1-203, Furthermore, we found that residues 1-44, 5' to the template region (residues 46-56) are not essential for activity, indicating a minimal functional region is located between residues 44-203, Mutagenesis of full-length hTR between residues 170-179, 180-189 or 190-199 almost completely abolished the ability of the hTR to function in the reconstitution of telomerase activity, suggesting that sequences or structures within this 30 nucleotide region are required for activity, perhaps by binding telomerase protein components.