Unusual basis of tRNA identity in human mitochondria

人类线粒体中 tRNA 身份的不寻常基础

• 批准号: 7455471
• 负责人: JOSEPH W CHIHADE
• 金额: $ 1.45万
• 依托单位: CARLETON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7455471
• 关键词: Alanine; Alanine-Specific tRNA; Alanine-tRNA Ligase; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Animals; Antibiotics; Bacteria; Be++ element; Beryllium; Biological Assay; C-terminal; Charge; Class; Defect; Development; Elements; Enzymes; Eukaryota; Eukaryotic Cell; Evolution; Facility Construction Funding Category; Family; Homologous Gene; Human; Hybrids; Individual; Kinetics; Lead; Ligase; Maps; Measures; Mitochondria; Mitochondrial Diseases; Mutagenesis; Mutation; Nucleotides; Organism; Peptides; Personal Satisfaction; Preparation; Property; Protein Region; Proteins; RNA; RNA Binding; RNA Recognition Motif; Rate; Research; Research Personnel; Role; Sequence Alignment; Side; Site; Site-Directed Mutagenesis; Source; Specificity; Structure; Substrate Specificity; System; Testing; Transfer RNA; Work; base; deletion analysis; interest; mutant; novel; nucleotide analog; programs; research study; species difference; stem; tRNA-alanine complex

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.

项目摘要。后生线粒体含有高度分歧的TRNA 序列和结构来自进化中其他地方的序列和结构。这些trnas被认可，并且 通过核编码的线粒体氨基酰基-TRNA专门为其同源氨基酸充电 合成酶。尽管这些酶与细胞质和细菌具有高度的序列相似性 对应物，对其异常tRNA底物的检查表明特定RNA的新型模式 可以使用认可。多宗线粒体丙苯甲酰-TRNA酶（ALARS）是特殊的 兴趣是因为在所有其他系统中定义丙氨酸身份的tRNA受体茎序列是 动物线粒体中的变化极大。 这里提出的研究的目的是阐明人类的特定适应 线粒体质量使该酶识别其奇异的tRNA底物。我们的初步结果 显示了来自细菌，真核生物和 其他后生线粒体可预测人线粒体酶的线粒体。 人线粒体系统中底物特异性的基础将通过映射tRNA确定 使用三种方法的身份元素：构建混合trnas，跨物种的氨基酰化 其他动物线粒体TRNA和核苷酸模拟干扰映射。这些实验会 指导制备突变体以评估单个核苷酸的重要性。 该研究还将确定导致改变的线粒体酶的适应 使用缺失分析的特异性来确定蛋白质的哪些保守RNA结合结构域 参与特定认可。定点突变和域掉期将用于检查 特定侧链或肽的作用。 关联。因为氨基酰基-TRNA合成酶是具有交叉的酶家族 物种在特异性方面的差异是抗生素发育的有吸引力的靶标。 AARS中的缺陷 对tRNA的识别也参与了几种遗传性线粒体疾病。这项工作将扩大我们的 对这个重要酶类的演变和特异性的基本理解，一种理解是 对与健康相关的应用的持续发展至关重要。