Unusual basis of tRNA identity in human mitochondria

项目总结。后生动物线粒体包含的tRNA在两种动物中都高度分化 序列和结构与进化中其他地方发现的序列和结构不同。这些tRNA被识别并 线粒体氨酰tRNA核糖核酸特异性携带同源氨基酸 合成酶。尽管这些酶与细胞质和细菌有很高的序列相似性 对应者，对其不同寻常的tRNA底物的研究表明，特定RNA的新模式 可以采用承认的方式。后生动物线粒体丙氨酰-tRNA合成酶(AlaRSs)具有特殊的功能 感兴趣的是，在所有其他系统中定义丙氨酸同一性的tRNA受体茎序列是 动物线粒体中的极易变的。 在这里提出的研究的目的是阐明人类的特定适应 线粒体AlaRs，使这种酶能够识别其奇怪的tRNA底物。我们的初步结果 展示以前鉴定的细菌、真核生物和 其他后生动物的线粒体并不能很好地预测人类的线粒体酶。 人类线粒体系统底物特异性的基础将通过对trna的定位来确定。 使用三种方法识别元件：杂合tRNA的构建，跨物种的氨酰化 其他动物线粒体tRNAs和核苷酸类似干扰图谱。这些实验将 指导突变体的准备，以评估单个核苷酸的重要性。 这项研究还将确定导致改变的线粒体酶的适应性 使用缺失分析来确定蛋白质的哪些保守的RNA结合域 涉及到特定的识别。将使用定点突变和结构域交换来检查 特定侧链或多肽的作用。 关联性。因为氨酰-tRNA合成酶是一个重要的酶家族，具有交叉. 由于物种特异性的差异，它们成为抗生素开发的有吸引力的目标。AARs中的缺陷 TRNA的识别也参与了几种可遗传的线粒体疾病。这项工作将扩展我们的 对这一重要酶类的进化和特异性的基本理解，即 这对健康相关应用的持续发展至关重要。

PROJECT SUMMARY. Metazoan mitochondria contain tRNAs which are highly diverged in both sequence and structure from those found elsewhere in evolution. These tRNAs are recognized and specifically charged with their cognate amino acids by nuclearly encoded mitochondrial aminoacyl-tRNA synthetases. Although the enzymes have a high degree of sequence similarity to cytoplasmic and bacterial counterparts, examination of their unusual tRNA substrates suggests that novel modes of specific RNA recognition may be employed. Metazoan mitochondrial alanyl-tRNA sythetases (AlaRSs) are of special interest because the tRNA acceptor stem sequence that defines alanine identity in all other systems is extremely variable in animal mitochondria. The objective of the research proposed here is to elucidate the specific adaptations of human mitochondrial AlaRS that allow this enzyme to recognize its bizarre tRNA substrate. Our preliminary results show RNA recognition properties of previously characterized homologues from bacteria, eukaryotes and other metazoan mitochondria poorly predict those of the human mitochondrial enzyme. The basis of substrate specificity in the human mitochondrial system will be determined by mapping tRNA identity elements using three approaches: construction of hybrid tRNAs, cross-species aminoacylation of other animal mitochondrial tRNAs, and nucleotide analog interference mapping. These experiments will guide the preparation of mutants to assess the importance of individual nucleotides. The research will also determine the adaptations in the mitochondrial enzyme responsible for altered specificity using deletion analysis to establish which of the conserved RNA binding domains of the protein are involved in specific recognition. Site-directed mutation and domain swaps will be used to examine the roles of specific side chains or peptides. RELEVANCE. Because aminoacyl-tRNA synthetases are an essential family of enzymes with cross- species differences in specificity, they make attractive targets for antibiotic development. Defects in aaRS recognition of tRNA are also involved in several inheritible mitochondrial diseases. This work will extend our basic understanding of the evolution and specificity of this important enzyme class, an understanding that is vital to the continuing development of health-related applications.