A Ty1 reverse transcriptase active-site aspartate mutation blocks transposition but not polymerization.
A Ty1 reverse transcriptase active-site aspartate mutation blocks transposition but not polymerization.
复制标题
Ty1 逆转录酶活性位点天冬氨酸突变会阻断转座，但不会阻断聚合。
DOI：
10.1128/jvi.75.14.6337-6347.2001
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发表时间：
2001
影响因子：
5.4
通讯作者：
Gabriel,A
中科院分区：
文献类型：
--
作者：
Uzun,O;Gabriel,A
逆转录酶 (RT) 存在于多种可移动遗传元件中，包括病毒、逆转录转座子和感染性细胞器内含子。不变的天冬氨酸三联体被认为是 RT 催化功能所必需的。我们在酵母反转录转座子 Ty1 中产生了 RT 突变体，将每个活性位点天冬氨酸改变为天冬酰胺或谷氨酸。除一种突变体外，所有突变体都缺乏可检测到的聚合酶活性。新的例外，D211N，在病毒样颗粒内保留了接近野生型的体外聚合酶活性，但未能进行体内转座。对于该突变体，负链合成受到损害，正链强终止中间体的形成被消除。转座缺陷的基因内第二位点抑制突变映射到该酶的 RNase H 结构域。我们的结果表明，逆转录转座子 RT 中的三个活性位点天冬氨酸之一并不具有催化关键性。这意味着 Ty1 和人类免疫缺陷病毒 RT 的聚合酶活性位点几何结构存在基本差异，并表明一个结构域中的细微突变可能会对同一酶的远处结构域造成显着的功能影响。
Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns. An invariant triad of aspartates is thought to be required for the catalytic function of RTs. We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate. All but one of the mutants lacked detectable polymerase activity. The novel exception, D211N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition. For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated. Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme.