Reverse transcriptase motifs in the catalytic subunit of telomerase
Reverse transcriptase motifs in the catalytic subunit of telomerase
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DOI：
10.1126/science.276.5312.561
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发表时间：
1997-04-25
期刊：
影响因子：
56.9
通讯作者：
Cech, TR
中科院分区：
文献类型：
--
作者：
Lingner, J;Hughes, TR;Cech, TR
端粒酶是大多数真核生物染色体末端复制所必需的核糖核蛋白酶。端粒酶RNA成分已经从许多生物体中鉴定出来，但没有蛋白质成分被证明能催化端粒DNA延伸。利用纳米电喷雾串联质谱法对端粒酶进行了纯化，并对其中一个蛋白进行了部分测序。该基因的克隆和序列分析表明，该123千道尔顿蛋白（p123）含有逆转录酶基序。一种酵母（酿酒酵母）的同源物被发现并随后被鉴定为EST2（更短的端粒），其缺失已被独立地证明会产生端粒缺陷。在酵母中，在Est2蛋白的逆转录酶基序中引入单氨基酸取代导致端粒缩短和衰老，这表明这些基序在体内催化端粒延长是重要的。野生型酵母菌提取物可以延长端粒DNA，而est2突变体或端粒酶RNA缺失突变体提取物则不会延长端粒DNA。因此，逆转录酶蛋白折叠，先前已知参与逆转录病毒复制和逆转录转位，是多种真核生物正常染色体端粒复制所必需的。
Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.