Pathways to Tertiary Structure Interfaces in RNA: An Integrated NMR, Biochemical, and Computational Approach

RNA 中三级结构界面的途径：综合 NMR、生化和计算方法

• 批准号: 2018296
• 负责人: Charles Hoogstraten
• 金额: $ 88万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2018296&HistoricalAwards=false
• 关键词: Pathways; Tertiary; Structure; Interfaces; RNA

RNA, a close chemical cousin of DNA, is most familiar in its messenger role of conveying information from the DNA genome to the cellular machinery that synthesizes proteins encoded by that genome. In recent decades, however, a great variety of noncoding roles for RNA molecules have been discovered, including in catalyzing chemical reactions, regulating gene expression, and in specialized functions such as CRISPR. To fulfill these roles, RNA takes up a wide variety of specific three-dimensional shapes and forms. Relatively little is known, however, about the formation of these three-dimensional RNA structures, leaving large gaps in the search for a predictive framework for the design of functional RNA molecules and of other molecules that target cellular RNAs. This project will carry out an integrated program of spectroscopic, biophysical, biochemical, and computational analyses in order to map out the energetics and folding pathways for RNA structures. The work will provide a full atomic-level description of the formation of an RNA three-dimensional structural interaction, and a scientific understanding of such RNA-RNA interactions in noncoding systems in general. This proposal will train underrepresented groups at the graduate and undergraduate levels. The PI’s efforts in mental-health awareness in the scientific workforce at regional and national conferences will be expanded.Although the factors underlying secondary structure (helix formation) in DNA and RNA are relatively well understood, the detailed study of the driving forces and mechanisms of formation of complex RNA tertiary structures has not kept pace with the great recent advances in RNA structural biology. The docking transition in the hairpin ribozyme is a rare example of a tertiary-only interaction amenable to intermolecular biophysical and spectroscopic investigation, with a wealth of existing structural and functional data and multiple functional readouts. This project will use a combination of NMR spectroscopy and molecular dynamics computations to examine conformational sampling of the individual loops and use functional assays of dynamically-quenched variants to determine the relevance of the observed motional modes determined in each loop and derive internally-consistent models of docking-competent states. An innovative protocol of NMR and Activity-initialized Iterative Markov Analysis (NAIMA) will be used to integrate all of the data in a computational determination of the pathway for the overall docking process. In the course of this work, the derivation of a reference database of unfolded RNA NMR chemical shifts for the general use of the field will be completed. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

RNA是DNA的化学近亲，它最为人所熟知的是它的信使角色，将信息从DNA基因组传递给细胞机器，细胞机器合成由基因组编码的蛋白质。然而，近几十年来，人们发现了RNA分子的多种非编码作用，包括催化化学反应、调节基因表达以及CRISPR等特殊功能。为了完成这些功能，RNA采用了多种特定的三维形状和形式。然而，人们对这些三维RNA结构的形成所知相对较少，这给设计功能性RNA分子和靶向细胞RNA的其他分子的预测框架的研究留下了很大的空白。该项目将进行光谱、生物物理、生化和计算分析的综合计划，以绘制RNA结构的能量学和折叠途径。这项工作将为RNA三维结构相互作用的形成提供一个完整的原子水平描述，并在一般的非编码系统中对这种RNA-RNA相互作用有一个科学的理解。这项建议将在研究生和本科两级培训代表性不足的群体。PI将在区域和国家会议上扩大在科学工作者中提高心理健康意识的努力。虽然DNA和RNA的二级结构（螺旋形成）的潜在因素已经被很好地理解，但对复杂RNA三级结构形成的驱动力和机制的详细研究还没有跟上RNA结构生物学近年来的巨大进展。发夹核酶的对接转变是一个罕见的三级相互作用的例子，适合分子间生物物理和光谱研究，具有丰富的现有结构和功能数据以及多个功能读数。该项目将结合核磁共振波谱和分子动力学计算来检查单个环的构象采样，并使用动态淬火变体的功能分析来确定每个环中观察到的运动模式的相关性，并推导出对接状态的内部一致模型。一种创新的核磁共振和活性初始化迭代马尔可夫分析（NAIMA）协议将用于整合所有数据，以计算确定整个对接过程的路径。在这项工作的过程中，将完成一个用于该领域普遍使用的未折叠RNA核磁共振化学位移参考数据库的推导。该项目得到了生物科学理事会分子和细胞生物科学部分子生物物理组的支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Spectroscopic characterization of Mn2+ and Cd2+ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme

锤头核酶保守 A9 金属位点中 Mn2 和 Cd2 与硫代磷酸酯配位的光谱表征

• DOI: 10.1016/j.jinorgbio.2022.111754
• 发表时间: 2022
• 期刊: Journal of Inorganic Biochemistry
• 影响因子: 3.9
• 作者: Hunsicker-Wang, Laura M.;Vogt, Matthew J.;Hoogstraten, Charles G.;Cosper, Nathaniel J.;Davenport, Audrey M.;Hendon, Christopher H.;Scott, Robert A.;Britt, R. David;DeRose, Victoria J.
• 通讯作者: DeRose, Victoria J.

Dynamics-Function Analysis in Catalytic RNA Using NMR Spin Relaxation and Conformationally Restricted Nucleotides

使用 NMR 自旋弛豫和构象限制性核苷酸对催化 RNA 进行动力学功能分析

• DOI: 10.1007/978-1-0716-0716-9_11
• 发表时间: 2021
• 期刊: Methods in molecular biology
• 作者: Hoogstraten, Charles G.;Terrazas, Montserrat;Aviñó, Anna;White, Neil A.;Sumita, Minako
• 通讯作者: Sumita, Minako