Structural characterization of gamma-glutamyl transferase enzymes

γ-谷氨酰转移酶的结构表征

• 批准号: 8465593
• 负责人: MARIE H HANIGAN
• 金额: $ 24.2万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8465593
• 关键词: 3-Dimensional; Applications Grants; Back; Base Pair Mismatch; Base Pairing; Base Sequence; Binding; Biochemical; Biological Assay; Centers of Research Excellence; Complex; Core Facility; Crystallization; Data; Development; Double-Stranded RNA; Drug Delivery Systems; Drug Design; Environment; Faculty; Goals; Guide RNA; Human; Immigrant; Individual; Infection; Instruction; Kinetics; Knowledge; Lead; Leishmania; Major Groove; Measures; Mentors; Messenger RNA; Mitochondria; Nucleotides; Oklahoma; Parasites; Pathway interactions; Patients; Play; Production; Proteins; Purines; RNA; RNA Editing; RNA Folding; RNA Interference; Reaction; Research Project Grants; Resolution; Ribonucleoproteins; Roentgen Rays; Role; Shapes; Site; Small RNA; Structure; System; Tail; Testing; Thermodynamics; Transcript; Trypanosoma; Trypanosoma brucei brucei; United States; United States National Institutes of Health; Variant; Width; X-Ray Crystallography; base; design; improved; instrument; interest; killings; mRNA Precursor; member; mutant; novel; purine; research study; simulation; structural biology; uridylate

Nonstandard base pairs in RNA often play important roles in RNA structure and function. These non- Watson/Crick base pairs are abundant in the thousands of guide RNA:mRNA complexes in the unique and essential RNA editing system of trypanosomes. The role of these nonstandard base pairs is unclear but is essential to understand for the design of drugs that target these RNAs, stop RNA editing, and kill trypanosomes. Our central hypothesis is that the effect of nonstandard base pairs on RNA editing, stability, and structure in these RNA complexes is dependent on their nucleotide sequence context. In specific aim 1, we test the effects of nonstandard base pairs on RNA editing efficiency with designed RNA editing substrates. Those substrates that lead to interesting effects on RNA editing will be selected for further study in specific aims 2 and 3. In aim 2, we will determine the contribution of nonstandard base pairs to thermodynamic stability and relate this knowledge back to RNA editing efficiency. In aim 3, we will determine the impacts of nonstandard base pairs on the overall shape ofthe RNA at low resolution (10-20 A) with small angle X-ray scattering and around the nonstandard base pair at high resolution (better than 2.5 A) using X-ray crystallography. The structural information will be interpreted in terms ofthe biochemical and thermodynamic data from aims 1 and 2. The pursuit of aim 3 will be greatly enhanced by the Oklahoma COBRE in Structural Biology through collaborative interactions with other COBRE and OSBN members, by mentoring from senior faculty members, and by access to COBRE Core Facilities. The results of this project will contribute to the development of better therapies to treat debilitating and often fatal infections with trypanosomal parasites, which infect 500,000 immigrants in the United States and threaten 600 million people worldwide.

RNA 中的非标准碱基对通常在 RNA 结构和功能中发挥重要作用。这些非 Watson/Crick 碱基对在数千个引导 RNA:mRNA 复合物中含量丰富，且独特且 锥虫的重要RNA编辑系统。这些非标准碱基对的作用尚不清楚，但 对于设计针对这些 RNA、停止 RNA 编辑和杀死 RNA 的药物至关重要 锥虫。我们的中心假设是非标准碱基对对 RNA 编辑、稳定性、 这些 RNA 复合物的结构取决于它们的核苷酸序列背景。在具体目标1中， 我们通过设计的RNA编辑测试非标准碱基对对RNA编辑效率的影响 基材。那些对 RNA 编辑产生有趣影响的底物将被选择进行进一步研究 在具体目标 2 和 3 中。在目标 2 中，我们将确定非标准碱基对对 热力学稳定性并将这些知识与 RNA 编辑效率联系起来。在目标 3 中，我们将 确定低分辨率（10-20）非标准碱基对对 RNA 整体形状的影响 A) 具有小角度 X 射线散射并以高分辨率（优于 2.5）围绕非标准碱基对 A) 使用X射线晶体学。结构信息将根据生化和 来自目标 1 和 2 的热力学数据。俄克拉荷马州将大大加强对目标 3 的追求 通过与其他 COBRE 和 OSBN 成员的协作互动，COBRE 在结构生物学中的应用 高级教职人员的指导，以及使用 COBRE 核心设施的机会。该项目的成果 将有助于开发更好的疗法来治疗使人衰弱且往往致命的感染 锥虫寄生虫，感染美国 50 万移民并威胁 6 亿人 世界各地的人们。