RNA folding and catalysis at the interface of biophysics and genomics

摘要 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。一是 一是严格的核酶机制，二是以发现为基础的RNA结构基因组学。这样做的一个主要目标是 建议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。 这一建议提出了一套关于RNA折叠和催化的可检验假说，并提出了新的 能够发现新的RNA生物学的技术。机械论策略的守恒提出了一些方法 其中，小核酶的自我切割被两种独特的催化策略激活。这些战略导致了 2‘羟基氢键状态的特殊变化可能降低其pKa以促进去质子化。 动力学实验、pKA测量和计算将在不同的 一组小的核酶，以及与机械相关的蛋白质酶。此外，CryoEM方法可以 解决小的、未修饰的核酶的结构涉及纳米对象结构的制备和快速 玻璃化将成为先驱。基于小核酶开发的冷冻EM技术将应用于小分子核酶 RNA和RNP复合体，以扩大低温EM对RNA领域的影响。为第三方提供的证据 提出了在核酶中缓冲催化的催化策略，并提出了一系列实验来验证这一点。 缓冲催化可以帮助解释各种RNA酶是如何工作的，包括大的核酶。我们还在寻求 为了了解体内的RNA折叠。将开发测量RNA折叠预测规则的方法 在类活体和活体条件下。将开发复杂的人造细胞质并用于测量结合 在荧光标记的RNA和它的未标记的补体之间。在这种混乱的系统中的滴定将是 作为温度的函数在qPCR板读取器上完成，以提供范特霍夫参数。 RNA折叠的预测规则也将在体内和基因组范围内测量，方法是将结构序列应用于 枯草芽孢杆菌在不同温度下生长。预测规则应改进RNA折叠预测 在活体条件下。RNA和蛋白质结构之间的反向关系将通过 测量选定蛋白质上的核糖体图谱。总体而言，机械和基因组之间的协同作用 将在多个层面上开发方法，并包括检测带电的开创性技术 为机械学研究和在全基因组范围内应用奠定了基础。此外，体内弱螯合的类镁离子 这些条件为研究RNA催化和折叠的刺激作用提供了理想的系统。最后，RNA 体内开发的预测规则将有助于描述催化RNA的折叠和功能。计算型 方法起着关键作用。它们用新的预测规则帮助从序列预测RNA结构， 帮助从结构描述符合作设计折叠RNA，并允许测试PKA降低 2‘-羟基亲核试剂。通过将实验室的生物物理学和基因组学方面结合起来，RNA生物学的新发现 将在分子水平上被制造和理解。

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.