Biological and biochemical functions of RNA in the Tetrahymena telomerase holoenzyme
Biological and biochemical functions of RNA in the Tetrahymena telomerase holoenzyme
复制标题
DOI：
10.1128/mcb.25.11.4442-4454.2005
复制
发表时间：
2005-06-01
影响因子：
5.3
通讯作者：
Collins, K
中科院分区：
文献类型：
--
作者：
Cunningham, DD;Collins, K
端粒酶通过合成串联简单序列重复来延伸染色体末端。对体外重建的最小重组端粒酶核糖核蛋白 (RNP) 的研究揭示了端粒酶 RNA 亚基 (TER) 内的序列，这些序列是建立其内部模板和酶活性的其他独特特征所必需的。在这里，我们测试了体内 TER 组装成端粒酶全酶后这些基序的重要性。我们建立了一种稳定表达表位标记的 TER 和 TER 变体代替野生型四膜虫 TER 的方法。我们发现TER非模板区域的序列替换改变了体内端粒长度的维持，端粒长度稳态的设定点增加或减少。我们还对细胞提取物中基于 RNA 的 RNP 亲和纯化后用 TER 变体重构的端粒酶全酶的体外活性进行了表征。我们发现非模板序列替换在模板使用的保真度和持续性方面造成了特定的缺陷。这些发现表明 TER 的非模板功能对于端粒酶全酶催化循环和体内适当的端粒长度维持至关重要。
Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.