Unusual basis of tRNA identity in human mitochondria

描述（由申请人提供）：后生动物线粒体含有trna，这些trna在序列和结构上与进化中其他地方发现的trna高度不同。这些trna被核编码的线粒体氨基酰基trna合成酶识别并特异性地携带其同源氨基酸。尽管这些酶与细胞质和细菌的对应物具有高度的序列相似性，但对它们不寻常的tRNA底物的研究表明，可能采用了新的特异性RNA识别模式。后生动物线粒体alanyl-tRNA合成酶（AlaRSs）之所以引起人们的特别关注，是因为在所有其他系统中定义丙氨酸身份的tRNA受体干序列在动物线粒体中是非常可变的。这里提出的研究目的是阐明人类线粒体AlaRS的特定适应性，使这种酶能够识别其奇异的tRNA底物。我们的初步结果表明，以前鉴定的细菌、真核生物和其他后生动物线粒体同源物的RNA识别特性很难预测人类线粒体酶的RNA识别特性。人类线粒体系统中底物特异性的基础将通过三种方法绘制tRNA识别元件来确定：杂交tRNA的构建、其他动物线粒体tRNA的跨物种氨基酰化和核苷酸类似物干扰作图。这些实验将指导突变体的制备，以评估单个核苷酸的重要性。该研究还将确定线粒体酶的适应性，使用删除分析来确定蛋白质的哪些保守RNA结合域参与特异性识别。位点定向突变和结构域交换将用于检查特定侧链或肽的作用。的相关性。由于氨基酰基trna合成酶是一个重要的酶家族，具有跨物种的特异性差异，它们成为抗生素开发的有吸引力的靶点。aaRS对tRNA的识别缺陷也与几种遗传性线粒体疾病有关。这项工作将扩展我们对这一重要酶类的进化和特异性的基本理解，这种理解对健康相关应用的持续发展至关重要。

DESCRIPTION (provided by applicant): 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 inheritable 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.