Zagryadskaya Ekaterina I, Kelley Shana O
Eugene F. Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Biochemistry. 2005 Jan 11;44(1):233-42. doi: 10.1021/bi0489560.
A series of disease-related mutations are known to affect the hs mt tRNA(Leu(UUR)) gene, and the molecular-level properties of this tRNA may underlie the effects of pathogenic sequence changes. A combinatorial approach has been used to explore the importance of the D, TPsiC, and anticodon loops of hs mt tRNA(Leu(UUR)) in the structure and function of this molecule. A tRNA library was constructed with 20 randomized nucleotides in the loop regions of hs mt tRNA(Leu(UUR)), and tRNA variants that were aminoacylated by hs mt LeuRS were isolated using an in vitro selection approach. Analysis of 26 selected sequences revealed that a stabilized anticodon stem significantly enhances aminoacylation activity. However, anticodon loop nucleotides were not conserved in the active sequences, indicating that this region of hs mt tRNA(Leu(UUR)) is not involved in recognition by LeuRS. Within the D and TPsiC loops, only two nucleotides conserved their identities, while new sequences were selected that likely mediate interloop interactions. The results indicate that hs mt tRNA(Leu(UUR)), which is known to have structurally weak D and anticodon stems, benefits functionally from the introduction of stabilizing interactions. However, the locations of individual nucleotides that govern discrimination of this tRNA by hs mt LeuRS still remain obscure.
已知一系列与疾病相关的突变会影响人源线粒体亮氨酸（UUR）转运RNA（hs mt tRNA(Leu(UUR))）基因，且该转运RNA的分子水平特性可能是致病序列变化产生影响的基础。已采用一种组合方法来探究人源线粒体亮氨酸（UUR）转运RNA的D环、TPsiC环和反密码子环在该分子结构和功能中的重要性。在人源线粒体亮氨酸（UUR）转运RNA的环区域构建了一个包含20个随机核苷酸的转运RNA文库，并使用体外筛选方法分离出了能被人源线粒体亮氨酰-tRNA合成酶（hs mt LeuRS）氨酰化的转运RNA变体。对26个选定序列的分析表明，稳定的反密码子茎显著增强了氨酰化活性。然而，反密码子环核苷酸在活性序列中并不保守，这表明人源线粒体亮氨酸（UUR）转运RNA的该区域不参与亮氨酰-tRNA合成酶的识别。在D环和TPsiC环内，只有两个核苷酸保持了它们的身份，同时还选择了可能介导环间相互作用的新序列。结果表明，已知具有结构上较弱的D环和反密码子茎的人源线粒体亮氨酸（UUR）转运RNA，在功能上受益于稳定相互作用的引入。然而，决定人源线粒体亮氨酰-tRNA合成酶对该转运RNA识别的单个核苷酸的位置仍然不清楚。
