Structure and Mechanism of Class II AA-tRNA Synthetases

II类AA-tRNA合成酶的结构和机制

• 批准号: 8072739
• 负责人: CHRISTOPHER S FRANCKLYN
• 金额: $ 33.93万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 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/8072739
• 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

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）：项目摘要氨基酰基TRNA合成酶（ARSS）催化氨基酰基tRNA的形成，以蛋白质合成和其他功能具有高效率和高精度。尽管进行了数十年的研究，但我们对反应机制，tRNA特异性的分子基础以及校对机理的理解是不完整的。在当前周期中，我们开发了快速的动力学方法来解决这些问题，并报告了I和II类ARSS和II类ARSS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出现作用以及对编辑的新见解。这些观察结果激发了描述整个II类ARS反应周期的综合模型的发展。为了充分验证该模型并解决了现场的其他长期问题，我们将（1）使用快速的动力学和生物物理技术将个体结合和化学步骤的速率与结构过渡相关联； （2）完成采用快速动力学，共振能量转移和其他生物物理技术的氨基酸编辑催化的氨基酸编辑的表征； （3）表征了代表性II类ARS的THRR的小分子抑制剂，从而在直接的生物医学背景下利用了我们的机械工作。到目前为止，尚未确定所有单个速率常数的综合动力学方案尚未报告任何编辑ARS。没有这样的最低方案，就无法正确评估转移前，转移后和取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。我们的研究将导致用于代表性编辑（THRR）和非编辑（HISRS）ARS的完整动力学方案，而我们为HISR定义的“触发器”机制可能是二聚体和四聚体ARS的一般特征。鉴于最近的数据涉及神经退行性疾病中的编辑缺陷，我们认为获取此详细的机械信息对于理解ARS结构/功能与真核蛋白质合成的复杂性质至关重要，这与神经生理学和疾病具有复杂的关系。我们的研究还将提供II类ARS抑制剂硼替肽的详细机械图，并提供进一步的生理数据，可用于鉴定诊断诊断诊断诊断诊断抗血管生成疗法的硼替肽衍生物。 公共卫生相关性：项目叙事氨基酰基-TRNA合成酶通过将氨基酸附加到其转移RNA衔接子中，在蛋白质合成中发挥重要作用。这些酶代表了针对社区获得的感染（即金黄色葡萄球菌）开发抗生素的验证靶标，并且在脑功能中具有其他复杂的作用，这些作用刚刚开始被欣赏。将来，针对这些酶的小分子调节剂可能会为减轻复杂神经疾病的新途径提供新的途径。