Roy Marc D, Wittenhagen Lisa M, Vozzella Brian E, Kelley Shana O
Boston College, Eugene F. Merkert Chemistry Center, Chestnut Hill, Massachusetts 02467, USA.
Biochemistry. 2004 Jan 20;43(2):384-92. doi: 10.1021/bi035711z.
The structure of the human mitochondrial (hs mt) tRNALeu(UUR) features several domains that are predicted to exhibit limited thermodynamic stability. An elevated frequency of disease-related mutations within these domains suggests a link between structural instability and the functional effects of pathogenic mutations. A series of tRNAs featuring mutations within the D and anticodon stems were prepared and investigated using nuclease probing. Structural mapping studies indicated that these domains were partially denatured for the wild type (WT) hs mt tRNALeu(UUR) and were significantly stabilized by mutations introducing additional or stronger base pairs into the stem regions. In addition, trends in the aminoacylation activities of the D stem mutants suggested that the loose structure is required for function, with mutants displaying the most ordered structures exhibiting the lowest levels of aminoacylation activity. A pronounced interdependence of the structures of the anticodon and D stems was observed, with mutations strengthening the D stem stabilizing the anticodon stem and vice versa. The existence of strong interdomain communication was further elucidated with a mutant of hs mt tRNALeu(UUR) containing a stabilized D stem and a pathogenic mutation that disrupted the anticodon stem. Strengthening the structure of the D stem completely restored the function of the disease-related mutant to WT levels, indicating that propagated structural weaknesses contribute to the functional deactivation of this tRNA by mutations.
人类线粒体（hs mt）亮氨酰-tRNA（UUR）的结构具有几个预测显示有限热力学稳定性的结构域。这些结构域内与疾病相关的突变频率升高，表明结构不稳定性与致病突变的功能效应之间存在联系。制备了一系列在D茎和反密码子茎内具有突变的tRNA，并使用核酸酶探测进行了研究。结构图谱研究表明，对于野生型（WT）hs mt亮氨酰-tRNA（UUR），这些结构域部分变性，并且通过在茎区域引入额外或更强碱基对的突变而显著稳定。此外，D茎突变体的氨酰化活性趋势表明，松散结构是功能所必需的，具有最有序结构的突变体表现出最低水平的氨酰化活性。观察到反密码子茎和D茎结构之间存在明显的相互依赖性，加强D茎的突变会稳定反密码子茎，反之亦然。通过一个含有稳定D茎和破坏反密码子茎的致病突变的hs mt亮氨酰-tRNA（UUR）突变体，进一步阐明了强结构域间通讯的存在。加强D茎的结构可将疾病相关突变体的功能完全恢复到野生型水平，表明传播的结构弱点导致该tRNA因突变而功能失活。
