Structure and Mechanism of Class II AA-tRNA Synthetases

II类AA-tRNA合成酶的结构和机制

基本信息

批准号：
7885744
负责人：
CHRISTOPHER S FRANCKLYN
金额：
$ 34.05万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-05-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7885744
关键词：
2-Aminopurine Abbreviations Active Sites Address Adenosine Alanine-tRNA Ligase Amino Acids Amino Acyl Transfer RNA Amino Acyl-tRNA Synthetases Aminoacylation Angiogenesis Inhibitors Animals Anti-Infective Agents Antibiotics Antineoplastic Agents Apoptosis Binding Biological Assay Brain Catalysis Cell physiology Chemicals Chemistry Chemotaxis Clinic Clinical Community-Acquired Infections Complement Complex Coumarins Coupled Cytosine Data Defect Development Diphosphates Disease Energy Transfer Enzymes Escherichia coli Event Fluorescence Resonance Energy Transfer Funding Future Gene Expression Glycine-tRNA Ligase Histidine-tRNA Ligase Human Hydrolysis Individual Investigation Kinetics Label Lead Ligase Link Mammalian Cell Measures Microarray Analysis Modeling Molecular Molecular Machines Molecular Motors Nature Neurodegenerative Disorders Pathway interactions Phosphate-Binding Proteins Physiological Physiology Polymerase Property Protein Biosynthesis RNA Reaction Relative (related person)Reporting Research Role Sampling Scheme Site Specificity Staphylococcus aureus Structure Techniques Testing Threonine Threonine-tRNA Ligase Toxic effect Transfer RNA Variant Work adenylate alpha benzopyrone analog antiangiogenesis therapy base chemical binding comparative cost helicase inhibitor/antagonist insight nervous system disorder proline-tRNA public health relevance relating to nervous system research study small molecule stopped-flow fluorescence tripolyphosphate

项目摘要

DESCRIPTION (provided by applicant): Project Summary Aminoacyl tRNA synthetases (ARSs) catalyze the formation of aminoacylated tRNA for protein synthesis and other functions with high efficiency and high accuracy. Despite decades of research, our understanding of the reaction mechanism, molecular basis of tRNA specificity, and mechanism of proofreading is incomplete. During the current cycle, we developed rapid kinetics approaches to address these questions, and reported a new functional distinction between class I and class II ARSs, two versions of substrate-assisted catalysis, a new emerging role for RNA in control of both reactions, and new insights into editing. These observations motivated the development of a comprehensive model that describes the entire class II ARS reaction cycle. To fully validate this model, and resolve other longstanding questions in the field, we will (1) correlate the rates of individual binding and chemical steps with structural transitions, employing rapid kinetics and biophysical techniques; (2) Complete the characterization of amino acid editing catalyzed by ThrRS and AlaRS, employing rapid kinetics, resonance energy transfer and other biophysical technique; and (3) characterize small molecule inhibitors of ThrRS, a representative class II ARS, thereby exploiingt our mechanistic work in a directly biomedical context. As yet, a comprehensive kinetic scheme in which all the individual rate constants have been determined has not been reported for any editing ARS. Without such a minimal scheme, one cannot properly assess the relative contributions of pre-transfer, post-transfer and re- sampling mechanisms to overall editing, nor properly calculate the physiological cost of editing. Our studies will result in complete kinetic schemes for representative editing (ThrRS) and non-editing (HisRS) ARSs, and the "flip-flop" mechanism we have defined for HisRS could be a general feature of dimeric and tetrameric ARSs. In view of recent data implicating editing defects in neurodegenerative diseases, we believe that acquiring this detailed mechanistic information is critical to understanding the linkage between ARS structure/function and the complex nature of eukaryotic protein synthesis, which has a complicated relationship to neural physiology and disease. Our studies will also provide a detailed mechanistic picture of a class II ARS inhibitor, borrelidin, and provide further physiological data useful in identifying borrelidin derivatives that are candidates for anti-angiogenic therapies in the clinic. PUBLIC HEALTH RELEVANCE: Project Narrative Aminoacyl-tRNA synthetases perform an essential function in protein synthesis by attaching amino acids to their transfer RNA adaptors. These enzymes represent validated targets for the development of antibiotics against community acquired infections (i.e., Staphylococcus aureus), and have other, complex roles in brain function that are just beginning to be appreciated. In the future, small molecule regulators directed against these enzymes may provide new avenues to alleviating complex neurological diseases.

描述（由申请人提供）：项目概要氨酰tRNA合成酶（ARS）催化氨酰化tRNA的形成，用于蛋白质合成和其他功能，具有高效率和高精度。尽管经过数十年的研究，我们对反应机制、tRNA 特异性的分子基础以及校对机制的理解并不完整。在当前周期中，我们开发了快速动力学方法来解决这些问题，并报告了 I 类和 II 类 ARS 之间的新功能区别、两种版本的底物辅助催化、RNA 在控制这两种反应中的新作用，以及对编辑的新见解。这些观察结果推动了描述整个 II 类 ARS 反应循环的综合模型的开发。为了充分验证该模型，并解决该领域其他长期存在的问题，我们将（1）采用快速动力学和生物物理技术，将单个结合和化学步骤的速率与结构转变相关联；（2）利用快速动力学、共振能量转移等生物物理技术，完成ThrRS和AlaRS催化氨基酸编辑的表征； (3)表征ThrRS（一种代表性的II类ARS）的小分子抑制剂，从而在直接生物医学背景下利用我们的机制工作。迄今为止，尚未报道任何编辑 ARS 的综合动力学方案，其中已确定所有单独的速率常数。如果没有这样一个最小的方案，人们就无法正确评估转移前、转移后和重采样机制对整体编辑的相对贡献，也无法正确计算编辑的生理成本。我们的研究将得出代表性编辑（ThrRS）和非编辑（HisRS）ARS的完整动力学方案，并且我们为HisRS定义的“触发器”机制可能是二聚体和四聚体ARS的一般特征。鉴于最近涉及神经退行性疾病编辑缺陷的数据，我们认为获取这种详细的机制信息对于理解 ARS 结构/功能与真核蛋白质合成的复杂性质之间的联系至关重要，真核蛋白质合成与神经生理学和疾病有着复杂的关系。我们的研究还将提供 II 类 ARS 抑制剂疏螺旋体素的详细机制图，并提供进一步的生理数据，用于识别作为临床抗血管生成疗法候选者的疏螺旋体素衍生物。公共健康相关性：项目叙述氨酰-tRNA 合成酶通过将氨基酸附着到其转移 RNA 接头上，在蛋白质合成中发挥重要功能。这些酶代表了开发针对社区获得性感染（即金黄色葡萄球菌）的抗生素的有效靶点，并且在大脑功能中具有其他复杂的作用，这些作用刚刚开始受到重视。未来，针对这些酶的小分子调节剂可能为缓解复杂的神经系统疾病提供新途径。