Unusual basis of tRNA identity in human mitochondria

描述(申请人提供)：后生动物线粒体包含的tRNA在序列和结构上都与进化中其他地方发现的tRNA有很大的差异。这些tRNA被核编码的线粒体氨基酰-tRNA合成酶识别并特异性地携带它们的同源氨基酸。虽然这些酶与细胞质和细菌中的对应酶具有高度的序列相似性，但对其不同寻常的tRNA底物的研究表明，可能会采用新的特定RNA识别模式。后生动物线粒体丙氨酰-tRNA合成酶(AlaRSs)具有特殊的意义，因为在动物线粒体中定义丙氨酸同一性的tRNA受体茎序列是非常不同的。这项研究的目的是阐明人类线粒体AlaRs的特殊适应，使该酶能够识别其奇怪的tRNA底物。我们的初步结果表明，以前描述的细菌、真核生物和其他后生动物线粒体同源物的RNA识别特性不能很好地预测人类线粒体酶的识别特性。人类线粒体系统底物特异性的基础将通过三种方法来确定：构建杂交tRNAs，其他动物线粒体tRNAs的跨物种氨酰化，以及核苷酸类似干扰作图。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。这项研究还将通过缺失分析来确定线粒体酶中负责特异性改变的适应性，以确定蛋白质的哪些保守的RNA结合域参与特异性识别。定点突变和结构域交换将被用来检测特定侧链或多肽的作用。关联性。由于氨基酰-tRNA合成酶是一种重要的酶家族，具有跨物种的特异性差异，因此它们是抗生素开发的有吸引力的靶点。TRNA的AARs识别缺陷也与几种遗传性线粒体疾病有关。这项工作将扩大我们对这一重要酶类的进化和特异性的基本理解，这一理解对于继续开发与健康相关的应用至关重要。

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.