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中的非标准碱基对在RNA的结构和功能中起着重要的作用。这些非- 沃森/克里克碱基对丰富的成千上万的指导RNA：mRNA复合物中的独特和 锥虫的基本RNA编辑系统。这些非标准碱基对的作用尚不清楚， 对于设计靶向这些RNA、停止RNA编辑和杀死 锥虫我们的中心假设是，非标准碱基对RNA编辑，稳定性， 并且这些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， 我们用设计的RNA编辑测试了非标准碱基对RNA编辑效率的影响， 印刷受体.将选择那些对RNA编辑产生有趣影响的底物进行进一步研究 具体目标2和3。在目标2中，我们将确定非标准碱基对对 热力学稳定性，并将这一知识与RNA编辑效率联系起来。在目标3中，我们 在低分辨率（10-20）下确定非标准碱基对RNA整体形状的影响 A）具有小角度X射线散射和高分辨率（优于2.5）的非标准碱基对周围 A）使用X射线晶体学。结构信息将被解释为生物化学和 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， 从资深教员的指导，并通过访问COBRE核心设施。该项目的结果 将有助于开发更好的治疗方法来治疗衰弱和往往致命的感染， 锥虫寄生虫，感染美国50万移民，威胁6亿人 世界各地的人们。