Structural characterization of gamma-glutamyl transferase enzymes

γ-谷氨酰转移酶的结构表征

• 批准号: 8666007
• 负责人: MARIE H HANIGAN
• 金额: $ 23.84万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8666007
• 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 Design; Drug Targeting; 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中的非标准碱基对往往在RNA的结构和功能中起着重要作用。这些非- Watson/Crick碱基对丰富，在成千上万的引导RNA：mRNA复合体中独一无二 锥虫基本RNA编辑系统。这些非标准碱基对的作用尚不清楚，但 对于设计以这些RNA为靶点的药物、停止RNA编辑和杀死 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复合体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编辑来测试非标准碱基对对RNA编辑效率的影响 底物。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 在目标2和目标3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，我们将 确定非标准碱基对在低分辨率(10-20)下对RNA整体形状的影响 A)在非标准碱基对周围有小角X射线散射，分辨率高(优于2.5 A)使用X射线结晶学。结构信息将被解释为生化和 来自目标1和目标2的热力学数据。俄克拉荷马州将极大地加强对目标3的追求 通过与其他Cobre和OSBN成员的协作互动，Cobre在结构生物学方面的研究 来自资深教职员工的指导，以及访问Cobre Core设施。这个项目的成果 将有助于开发更好的疗法来治疗衰弱的、往往是致命的感染 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。