A molecular switch underlies a human telomerase disease
A molecular switch underlies a human telomerase disease
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DOI：
10.1073/pnas.262663599
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发表时间：
2002-12-24
影响因子：
11.1
通讯作者：
James, TL
中科院分区：
文献类型：
--
作者：
Comolli, LR;Smirnov, I;James, TL
端粒酶是维持端粒所必需的核糖核蛋白（RNP）。虽然上调的端粒酶活性与晚期癌变的细胞永生特征密切相关，但最近，人类端粒酶RNA基因的突变与先天性角化不良和再生障碍性贫血相关，两者均以造血功能受损为特征。这些突变包括高度保守的推定端粒酶RNA假结中的碱基变化。在这里，通过使用体外端粒酶测定，NMR和UV吸光度熔融分析的模型寡核苷酸设计形成一个“反式假结”，我们描述的功能，结构和能量特性的这种结构。我们证明了pseucloknot域存在于两个替代状态的几乎相等的稳定性的解决方案：一个是先前提出的pseudokont配对P3与循环域P2b，另一个是一个结构化的P2b循环单独形成。我们发现，在一个先天性唇角化症家族中，一个基因拷贝中存在的两个碱基突变（GC 107/8 --> AG）使端粒酶活性丧失。该突变使P2b内环结构超稳定，阻断假结形成。相反，当P3假结配对通过删除P3中的保守凸起而超稳定时，端粒酶活性也降低。我们认为P2b/P3假纽结结构域作为一个分子开关，其两种状态之间的相互转换对端粒酶的功能是重要的。35个物种的P2b和P3序列的系统发育共变提供了一组引人注目的“自然”补偿碱基配对变化，支持关键分子开关的存在。
Telomerase is a ribonucleoprotein (RNP) required for maintenance of telomeres. Although up-regulated telomerase activity is closely linked to the cellular immortality characteristic of late stage carcinogenesis, recently, mutations in the telomerase RNA gene in humans have been associated with dyskeratosis congenital and aplastic anemia, both typified by impaired haemopoletic function. These mutations include base changes in a highly conserved putative telomerase RNA pseudoknot. Here, by using in vitro telomerase assays, NMR, and UV absorbance melting analyses of model oligonucleotides designed to form a "trans-pseudoknot," we describe functional, structural, and energetic properties of this structure. We demonstrate that the pseucloknot domain exists in two alternative states of nearly equal stability in solution: one is the previously proposed pseudoknot formed by pairing P3 with the loop domain of P2b, and the other is a structured P2b loop alone. We show that the two-base mutation (GC107/8 --> AG) present in one gene copy in a family with clyskeratosis congenital abrogates telomerase activity. This mutation hyperstabilizes the P2b intra-loop structure, blocking pseucloknot formation. Conversely, when the P3 pseucloknot pairing is hyperstabilized by deleting a conserved bulge in P3, telomerase activity also decreases. We propose that the P2b/P3 pseucloknot domain acts as a molecular switch, and interconversion between its two states is important for telomerase function. Phylogenetic covariation in the P2b and P3 sequences of 35 species provides a compelling set of "natural" compensatory base pairing changes supporting the existence of the crucial molecular switch.