Class II Aminoacyl-tRNA Synthetase Substrate Recognition

II 类氨酰基-tRNA 合成酶底物识别

• 批准号: 7752453
• 负责人: Karin M Musier-Forsyth
• 金额: $ 31.41万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7752453
• 关键词: Active Sites; Affinity Chromatography; Alanine; Amino Acid Sequence; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Antibiotics; Antifungal Agents; Architecture; Binding; Biological Assay; Catalysis; Catalytic Domain; Cell Culture Techniques; Cells; Complex; Cysteine; Development; Discrimination; Disease; Docking; Enzymes; Escherichia coli; Family; Family member; Genetic Code; Genome; Grant; Homologous Gene; Hydrolase; Hydrolysis; In Vitro; Kinetics; Ligase; Maps; Mutagenesis; Neurodegenerative Disorders; Pathway interactions; Peptide Sequence Determination; Process; Proline-Specific tRNA; Protein Biosynthesis; Protein Family; Proteins; Quality Control; Reaction; Relative (related person); Research; Research Design; Role; Screening procedure; Site; Site-Directed Mutagenesis; Specificity; Structure; Technology; Tertiary Protein Structure; Testing; Transfer RNA; Translations; Work; X-Ray Crystallography; adenylate; analytical ultracentrifugation; base; crosslink; gel electrophoresis; in vivo; insight; member; mutant; novel; prevent; proline-tRNA; public health relevance; stoichiometry

DESCRIPTION (provided by applicant): The overarching objective of this work is to contribute to our fundamental understanding of how high fidelity in protein synthesis is achieved during translation of the genetic code. Accurate substrate recognition and discrimination by aminoacyl-tRNA synthetases (aaRS) is a key feature of this process. There are two classes of aaRS, and research in the Musier-Forsyth lab for the past sixteen years has revolved around class II synthetases, with a strong focus on prolyl-tRNA synthetase (ProRS). During the previous grant period, we began turning our focus to quality control mechanisms that prevent mistranslation due to the misactivation of structurally related amino acids by aaRSs. After misactivation, some noncognate amino acids are immediately hydrolyzed in the aminoacylation active site (i.e., the first or "coarse sieve") in a reaction known as "pre- transfer" editing; others are attached to tRNA and translocated to an editing site that has been designated the second or "fine sieve". The latter domain carries out a reaction referred to as "post-transfer" editing. ProRSs display an unusually diverse range of domain architectures. Bacterial enzymes contain an editing domain (INS) that is responsible for clearing misacylated Ala-tRNAPro. The INS domain is missing from most archaeal and eukaryotic enzymes, however, genome-encoded single-domain proteins (e.g., YbaK family) with homology to the ProRS INS domain are widely distributed in all three kingdoms. Interestingly, some members of this family have been shown to possess Cys-tRNAPro editing activity and to interact with the ProRS/tRNA complex. These observations allowed us to propose a novel "triple-sieve" mechanism of editing, which forms the basis of the present renewal application. Here, we propose to continue to explore the specificities and novel functions of these intriguing and poorly understood single-domain proofreading proteins, as well as their mechanisms and species-specific differences. The specific aims are: 1) To explore pre-transfer editing by ProRS, 2) to explore the post-transfer editing mechanism of ProRS and ProRS-like editing domains, 3) to explore the function of YbaK and homologous families of proteins in vitro and in vivo, and 4) to probe the structure of the ProRS/YbaK/tRNA ternary complex. PUBLIC HEALTH RELEVANCE: The objective of this work is to contribute to our fundamental understanding of how high fidelity in protein synthesis is achieved during translation of the genetic code. The aminoacyl-tRNA synthetases and related genome-encoded single domain proteins studied in this work, possess editing functions that prevent errors in protein synthesis. Editing-defective synthetases have been implicated in progressive neurodegenerative diseases. Thus, in recent years, it has become clear that a better understanding of the editing functions of synthetases may provide insights and novel treatments for various diseases. Due to their essential function, these enzymes are also promising targets for the development of antibiotics and antifungal agents.

描述（由申请人提供）：这项工作的总体目标是有助于我们对遗传密码翻译过程中如何实现蛋白质合成的高保真度的基本理解。氨基酰基-TRNA合成酶（AARS）的准确底物识别和歧视是该过程的关键特征。 AARS有两类，在过去的16年中，在Musier-Forsyth实验室中进行了研究，围绕II类合成酶进行了旋转，重点是丙酰 - TRNA合成酶（PRORS）。在上一个赠款期间，我们开始将重点转向质量控制机制，以防止由于AARS对结构相关的氨基酸误导而导致误译。误导后，在称为“预转移”编辑的反应中，在氨基酰化活性位点（即，第一个或“粗筛”）中立即水解了一些非认知氨基酸；其他人则附着在tRNA上，并转移到已被指定为第二个或“细筛”的编辑网站上。后一个域进行了一种称为“转移后”编辑的反应。 Prors展示了异常多样的域架构。细菌酶含有一个编辑域（INS），该酶负责清除降低的Ala-trnapro。但是，大多数古细菌和真核酶都缺少INS结构域，但是，基因组编码的单域蛋白（例如YBAK家族）与Prors INS域具有同源性，在所有三个王国中都广泛分布。有趣的是，该家族的一些成员已被证明具有Cys-trnapro编辑活动并与PROR/TRNA复合物相互作用。这些观察结果使我们能够提出一种新颖的“三重关系”编辑机制，这构成了当前更新应用的基础。在这里，我们建议继续探索这些有趣且知之甚少的单域校对蛋白的特异性和新功能，以及它们的机制和物种特定的差异。具体目的是：1）探索PRORS的前传输前编辑，2）探索传输后的编辑机制，类似于prors的编辑域，3）探索YBAK和蛋白质在体外和体外的蛋白质家族的功能，以及4）探测Prors/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak/ybak trolna nernary nornary nornary nornary sernary nornary sernary sernary sernary sernary sernary sernary sernary。 公共卫生相关性：这项工作的目的是为我们对在遗传密码翻译过程中对蛋白质合成的高忠诚的基本理解做出贡献。在这项工作中研究的氨基酰基-TRNA合成酶和相关的基因组编码的单域蛋白质具有编辑功能，可防止蛋白质合成中的错误。编辑缺陷性合成酶已与进行性神经退行性疾病有关。因此，近年来，很明显，对合成酶的编辑功能的更好理解可以为各种疾病提供见解和新颖的治疗方法。由于其必不可少的功能，这些酶也是开发抗生素和抗真菌剂的有希望的靶标。