RNA folding and catalysis at the interface of biophysics and genomics

生物物理学和基因组学交叉领域的 RNA 折叠和催化

• 批准号: 10394217
• 负责人: PHILIP C BEVILACQUA
• 金额: $ 38.13万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394217
• 关键词: Area; Bacillus subtilis; Binding; Biological Products; Biology; Biophysics; Buffers; Catalysis; Catalytic RNA; Charge; Chelating Agents; Complement; Complex; Cryoelectron Microscopy; Cytoplasm; Descriptor; Enzymes; Genomic approach; Genomics; Goals; Hydrogen Bonding; Kinetics; Label; Lead; Measurement; Measures; Methods; Molecular; Play; Preparation; Proteins; RNA; Reader; Structure; System; Techniques; Temperature; Testing; Therapeutic; Titrations; Work; base; deprotonation; design; driving force; experimental study; genome-wide; improved; in vivo; nano-objects; new technology; novel; protein structure; ribosome profiling; structural genomics; synergism

Abstract The Bevilacqua lab has contributed to the field of RNA biology through two disparate approaches. One is rigorous ribozyme mechanism and the other is discovery-based RNA structural genomics. A major goal of this proposal is to bridge these two areas to discover new RNA biology and to characterize it at the molecular level. This proposal advances a set of testable hypotheses on RNA folding and catalysis, as well as proposes new technologies to enable discovery of novel RNA biology. Conservation of mechanistic strategies suggests ways in which small ribozyme self-cleavage is activated by two unique catalytic strategies. These strategies lead to specific changes in the hydrogen bonding status of the 2′OH that could lower its pKa to facilitate deprotonation. Kinetics experiments, pKa measurements, and calculations will be implemented and conducted on a diverse set of small ribozymes, as well as mechanistically related protein enzymes. In addition, cryoEM approaches to solve structures of small, unmodified ribozymes involving preparation of nano-objects structures and rapid vitrification will be pioneered. Techniques for cryoEM developed on small ribozymes will be applied to small RNA and RNP complexes to expand the impact of cryoEM on the RNA field. Evidence is provided for a third catalytic strategy, which is buffer catalysis in ribozymes, and a suite of experiments is proposed to test this. Buffer catalysis could help explain how diverse RNA enzymes work including large ribozymes. We also seek to understand RNA folding in vivo. Methods will be developed to measure RNA folding prediction rules under in vivo-like and in vivo conditions. Complex artificial cytoplasms will be developed and used to measure binding between a fluorescently labeled RNA and its unlabeled complement. Titrations in such `messy systems' will be accomplished on a qPCR plate reader as a function of temperature to provide van't Hoff parameters. Prediction rules for RNA folding will also be measured in vivo and genome-wide by applying Structure-seq to Bacillus subtilis growing at different temperatures. The prediction rules should improve RNA folding prediction under in vivo conditions. An inverse relationship between RNA and protein structure will be pursued by measuring ribosome profiling on select proteins. Overall, synergy between mechanistic and genomic approaches will be developed on multiple levels and includes pioneering techniques for detecting charged bases for mechanistic studies and applying them genome wide. In addition, weakly chelated in vivo-like Mg2+ conditions provide an ideal system for investigating stimulation of RNA catalysis and folding. Finally, RNA prediction rules developed in vivo will help describe the folding and function of catalytic RNAs. Computational approaches play a key role. They aid prediction of RNA structure from sequence with new prediction rules, help design cooperatively folding RNAs from structural descriptors, and allow testing of pKa lowering of the 2′OH nucleophile. By uniting the biophysical and genomics aspect of the lab, new discoveries in RNA biology will be made and understood at the molecular level.

抽象的 Bevilacqua 实验室通过两种不同的方法为 RNA 生物学领域做出了贡献。 严格的核酶机制，另一个是基于发现的RNA结构基因组学的一个主要目标。 提案旨在将这两个领域联系起来，以发现新的 RNA 生物学并在分子水平上表征它。 该提案提出了一组关于 RNA 折叠和催化的可测试假设，并提出了新的 能够发现新型 RNA 生物学的技术提出了方法。 其中小核酶的自裂解是由两种独特的催化策略激活的。 2'OH 氢键状态的特定变化可以降低其 pKa 以促进去质子化。 动力学实验、pKa 测量和计算将在不同的平台上实施和进行 一组小核酶，以及机械相关的蛋白质酶此外，冷冻电镜方法。 解决小型未修饰核酶的结构 纳米物体结构的制备和快速 玻璃化冷冻技术将率先应用于小型核酶。 RNA 和 RNP 复合物扩大了冷冻电镜对 RNA 领域的影响，为第三个领域提供了证据。 催化策略，即核酶中的缓冲催化，并提出了一系列实验来测试这一策略。 缓冲液催化可以帮助解释包括大核酶在内的多种 RNA 酶的工作原理。 为了了解体内RNA折叠，将开发测量RNA折叠预测规则的方法。 将开发复杂的人工细胞质并用于测量结合。 在这种“混乱的系统”中，荧光标记的 RNA 与其未标记的互补体之间的滴定将是困难的。 在 qPCR 酶标仪上完成，作为温度的函数，以提供 vant Hoff 参数。 RNA 折叠的预测规则也将通过应用 Structure-seq 在体内和全基因组范围内进行测量 枯草芽孢杆菌在不同温度下生长的预测规则应该可以改善 RNA 折叠预测。 在体内条件下，RNA 和蛋白质结构之间的反比关系将由 总体而言，测量选定蛋白质的核糖体分析。机械和基因组之间的协同作用。 方法将在多个层面上开发，包括检测带电的开创性技术 此外，体内弱螯合的 Mg2+ 也为机制研究和全基因组应用奠定了基础。 条件为研究 RNA 催化和折叠的刺激提供了理想的系统。 体内开发的预测规则将有助于描述计算 RNA 的折叠和功能。 它们通过新的预测规则帮助预测 RNA 结构， 帮助根据结构描述符设计协同折叠 RNA，并允许测试 pKa 降低 2′OH亲核试剂通过结合实验室的生物物理和基因组学方面，RNA生物学的新发现。 将在分子水平上被制造和理解。