Mechanism of proofreading by class I aminoacyl-tRNA synthetases

I 类氨酰基-tRNA 合成酶的校对机制

• 批准号: 7500879
• 负责人: JOHN J. PERONA
• 金额: $ 3.26万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7500879
• 关键词: Active Sites; Address; Amino Acids; Amino Acyl Transfer RNA; Amino Acyl-tRNA Synthetases; Aminoacylation; Ammonia; Anti-Bacterial Agents; Architecture; Asparagine-Specific tRNA; Binding; Biochemical; Biological Assay; Biological Models; Catalysis; Catalytic Domain; Cells; Class; Complement; Complex; Coupled; Coupling; Crystallization; Cysteine-tRNA ligase; Data; Depth; Development; Discrimination; Distant; Electrostatics; Engineering; Ensure; Enzymes; Escherichia coli; Esters; Event; Evolution; Family; Fluorescence Spectroscopy; Foundations; Genetic; Glutamine; Glutamine-Specific tRNA; Glutamine-tRNA ligase; Goals; Homologous Gene; Human; Hydrolysis; Individual; Investigation; Kinetics; Laboratories; Life; Ligand Binding; Ligase; Location; Measurement; Measures; Methodology; Methods; Microscopic; Modeling; Molecular; Molecular Conformation; Monitor; Mutagenesis; Nature; Nucleotides; Numbers; One-Step dentin bonding system; Organism; Pathway interactions; Placement; Process; Protein Biosynthesis; Protein Engineering; Proteins; Public Health; Pylorus; Quality Control; RNA, Transfer, Amino Acid-Specific; Range; Rate; Reaction; Research; Resolution; Roentgen Rays; Role; Shapes; Signal Transduction; Site; Solvents; Specificity; Structure; Structure-Activity Relationship; System; Techniques; Thermodynamics; Transfer RNA; Valine-tRNA Ligase; Work; Zinc; adenylate; analog; antimicrobial drug; base; cost; deacylation; design; enzyme activity; enzyme structure; enzyme substrate; fluorophore; functional group; glutamine-tRNA; improved; inhibitor/antagonist; insight; interest; leucine-tRNA; mutant; novel; parent grant; pathogenic bacteria; research study; stopped-flow fluorescence; success; tool; transmission process

DESCRIPTION (provided by applicant): Aminoacyl-tRNA synthetases (aaRS) are the enzymes ensuring faithful transmission of genetic information in all living cells. They match cognate amino acids with tRNAs, forming an aminoacyl-tRNA ester by way of an aminoacyl-adenylate intermediate. Some tRNA synthetases cannot distinguish between structurally similar amino acids with high accuracy, and thus proceed in catalysis of the noncognate reaction. However, the noncognate aminoacyl-adenylate and aminoacyl-tRNA are destroyed by additional hydrolytic activities of the enzymes (pre- and post-transfer editing or proofreading, respectively). Hydrolysis of the noncognate aminoacylated tRNA (post-transfer editing) occurs in a spatially separate editing domain. Crystal structures support a model whereby the single stranded 3'-end of the aminoacyl-tRNA is translocated from the synthetic to the hydrolytic site some 30 E distant. Hydrolysis of the noncognate aminoacyl-adenylate (pre-transfer editing) has been assumed to occur at the same remote hydrolytic site and shuttling of the intermediate was therefore proposed. However, crystal structures have never corroborated the existence of the shuttling pathway. Recently, it was shown that hydrolysis of cognate glutaminyl-adenylate, an analogous reaction to pre-transfer editing, may occur in the confines of the active site of a nonediting class I glutaminyl-tRNA synthetase. This strongly suggests that hydrolysis of noncognate aminoacyl-adenylate can occur in the homologous synthetic sites of class I editing synthetases such as isoleucyl-, valyl- and leucyl-tRNA synthetases (IleRS, ValRS and LeuRS, respectively). The long-term objective of this project is to elucidate the mechanisms of the editing activities of IleRS and ValRS toward noncognate amino acids. Two specific aims (1 and 2) are proposed to address the following questions: (i) does pre-transfer editing require tRNA and if it does what is its role? (ii) where does the hydrolysis of noncognate aminoacyl-adenylate take place?; (iii) does amino acid discrimination operate through conformational readjustment of both synthetase and tRNA during aminoacylation and/or translocation step? Addressing these questions will also require the development of suitable methodology. A new aminoacyl-adenylate synthesis assay (recently developed for the study of GlnRS), as well as novel partitioning assays (proposed herein) will be applied to follow the fate of the aminoacyl-adenylate. tRNA analogues and post-transfer editing deficient protein mutants will be used in the experiments designed to evaluate a role for tRNA and location of pre-transfer editing. Stopped flow fluorescence will be pursued, using several different fluorophores, to explore conformational readjustment of synthetase and tRNA during the course of the noncognate and cognate reactions. The data obtained will significantly improve our understanding of amino acid discrimination by editing class I aminoacyl-tRNA synthetases. PUBLIC HEALTH RELEVANCE: Accurate aminoacyl-tRNA synthesis is a prerequisite to the survival of all cells. Consequently, the proofreading activities of aminoacyl-tRNA synthetases have been conserved through evolution in spite of the high energetic cost that the reactions impose on the cell. Understanding of the editing mechanism in detail (Specific Aims 1 and 2) would allow better use of these enzymes as targets for antibacterial agents, because the editing pathway represents an additional selective target for inhibitor action.

描述（由申请人提供）： 氨酰基-tRNA 合成酶（aaRS）是确保所有活细胞中遗传信息忠实传递的酶。它们将同源氨基酸与 tRNA 匹配，通过氨酰基-腺苷酸中间体形成氨酰基-tRNA 酯。一些tRNA合成酶不能高精度地区分结构相似的氨基酸，从而继续催化非同源反应。然而，非同源的氨酰基-腺苷酸和氨酰基-tRNA 会被酶的额外水解活性（分别是转移前和转移后编辑或校对）破坏。非同源氨酰化 tRNA 的水解（转移后编辑）发生在空间上独立的编辑域中。晶体结构支持这样一种模型，即氨酰基-tRNA 的单链 3' 端从合成位点易位到距离约 30 E 的水解位点。假定非同源氨酰基腺苷酸的水解（转移前编辑）发生在相同的远程水解位点，因此提出了中间体的穿梭。然而，晶体结构从未证实穿梭路径的存在。最近，研究表明，同源谷氨酰胺酰腺苷酸的水解（一种与预转移编辑类似的反应）可能发生在非编辑 I 类谷氨酰胺酰-tRNA 合成酶的活性位点范围内。这强烈表明非同源氨酰基-腺苷酸的水解可以发生在I类编辑合成酶的同源合成位点，例如异亮氨酰-、缬氨酰-和亮氨酰-tRNA合成酶（分别为IleRS、ValRS和LeuRS）。该项目的长期目标是阐明 IleRS 和 ValRS 对非同源氨基酸的编辑活性机制。提出了两个具体目标（1 和 2）来解决以下问题：(i) 转移前编辑是否需要 tRNA，如果需要，其作用是什么？ (ii) 非同源氨酰基腺苷酸的水解在哪里发生？ (iii) 氨基酸区分是否通过氨酰化和/或易位步骤期间合成酶和 tRNA 的构象重新调整来进行？解决这些问题还需要制定合适的方法。新的氨酰腺苷酸合成测定（最近为 GlnRS 研究而开发）以及新的分配测定（本文提出）将用于追踪氨酰腺苷酸的命运。 tRNA 类似物和转移后编辑缺陷蛋白突变体将用于旨在评估 tRNA 的作用和转移前编辑位置的实验。将使用几种不同的荧光团进行停流荧光，以探索非同源和同源反应过程中合成酶和 tRNA 的构象重新调整。获得的数据将显着提高我们对通过编辑 I 类氨酰基-tRNA 合成酶进行氨基酸区分的理解。公共卫生相关性：准确的氨酰基-tRNA 合成是所有细胞生存的先决条件。因此，尽管反应对细胞造成了很高的能量消耗，但氨酰-tRNA 合成酶的校对活性在进化过程中得到了保留。详细了解编辑机制（具体目标 1 和 2）将有助于更好地利用这些酶作为抗菌剂的靶点，因为编辑途径代表了抑制剂作用的另一个选择性靶点。