Aminoacylation properties of pathology-related human mitochondrial tRNALys variants
Aminoacylation properties of pathology-related human mitochondrial tRNALys variants
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DOI：
10.1261/rna.5267604
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发表时间：
2004-05-01
期刊：
影响因子：
4.5
通讯作者：
Florentz, C
中科院分区：
文献类型：
--
作者：
Sissler, M;Helm, M;Florentz, C
体外转录已被证明是制备功能性RNA的成功工具，特别是在tRNA领域，其中尽管不存在转录后修饰，但转录物正确折叠并具有功能活性。人类线粒体（mt）tRNA（Lys）偏离了这一原则，并折叠成各种无活性构象，这是由于缺乏转录后修饰m（1）A9，其阻碍了天然tRNA中与U64的碱基配对。功能性转录物的不可用是结构/功能研究以及破译mt tRNA（Lys）基因中的点突变引起严重人类疾病的分子机制的严重缺点。在这里，我们表明，工程在体外转录的“假WT”tRNA（赖氨酸）的变体是有效地识别赖氨酰-tRNA合成酶，并可以取代WT tRNA作为一个有价值的参考分子。这已被利用在迄今为止所描述的9种病理相关突变对氨酰化的影响的系统分析中。位于tRNA二级结构环中的唯一突变A8344 G不影响氨酰化效率。在位于螺旋结构域的八个突变中，将典型的Watson-Crick对转化为G-U对或C（.）A错配，六个对氨酰化没有影响（A8296 G，U8316 C，G8342 A，U8356 C，U8362 G，G8363 A），两个导致裂解效率急剧下降（5000- 7000倍）（G8313 A和G8328 A）。这种筛选，允许分析在可比条件下影响一种tRNA的所有突变的主要影响水平，表明不同疾病的不同分子起源。
In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA(Lys) deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m(1)A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA(Lys) gene cause severe human disorders. Here, we show that an engineered in vitro transcribed "pseudo-WT" tRNA(Lys) variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson-Crick pairs into G-U pairs or C(.)A mismatches, six have no effect on aminciacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000- to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders.