Autexier C, Triki I
Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research,The Sir Mortimer B. Davis-Jewish General Hospital, Montréal, Quebec H3T 1E2, Canada.
Nucleic Acids Res. 1999 May 15;27(10):2227-34. doi: 10.1093/nar/27.10.2227.
Telomerase is an enzyme that is essential for the replication and maintenance of chromosomal termini. It is a ribonucleoprotein consisting of a catalytic subunit, one or more associated proteins, and an integral RNA subunit that serves as a template for the synthesisof telomeric repeats. We identified a Tetrahymena telomerase RNA-protein complex by an electrophoretic mobility shift assay, using telomerase partially purified from whole cell extracts and radiolabeled, in vitro transcribed wild-type Tetrahymena telomerase RNA. Complex formation was specific as unlabeled Tetra-hymena telomerase RNA, but not Escherichia coli ribo-somal RNAs, competitively inhibited complex formation. Binding required concentrations of MgCl2of at least 10 mM and occurred over a wide range of potassium glutamate concentrations (20-220 mM). The RNA-protein complex was optimally reconstituted with a 30 degrees C preincubation for </=5 min, prior to electrophoresis. Certain Tetrahymena telomerase RNAs containing deletions of structures and sequences previously predicted to be involved in protein binding were unable to competitively and specifically inhibit complex formation, suggesting a role in protein binding for the deleted residues or structures.
端粒酶是一种对于染色体末端的复制和维持至关重要的酶。它是一种核糖核蛋白，由一个催化亚基、一种或多种相关蛋白质以及一个作为端粒重复序列合成模板的完整RNA亚基组成。我们通过电泳迁移率变动分析鉴定了一种四膜虫端粒酶RNA-蛋白质复合物，使用从全细胞提取物中部分纯化的端粒酶以及放射性标记的、体外转录的野生型四膜虫端粒酶RNA。复合物的形成具有特异性，因为未标记的四膜虫端粒酶RNA，而非大肠杆菌核糖体RNA，能竞争性抑制复合物的形成。结合需要至少10 mM的MgCl2浓度，并且在广泛的谷氨酸钾浓度范围（20 - 220 mM）内发生。在电泳前，RNA-蛋白质复合物通过在30℃预孵育≤5分钟能得到最佳重组。某些含有先前预测参与蛋白质结合的结构和序列缺失的四膜虫端粒酶RNA无法竞争性且特异性地抑制复合物的形成，这表明缺失的残基或结构在蛋白质结合中起作用。
