Collaborative Research: Uncovering How Riboswitches Exploit Out-of-Equilibrium RNA Folding Pathways to Make Genetic Decisions

合作研究：揭示核糖开关如何利用非平衡 RNA 折叠途径做出遗传决策

• 批准号: 1914567
• 负责人: Julius Lucks
• 金额: $ 35.16万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1914567&HistoricalAwards=false
• 关键词: Collaborative; Research; Uncovering; How; Riboswitches

This work will uncover new principles by which riboswitch RNAs exploit out-of-equilibrium RNA folding and ligand binding to make genetic decisions. The tools developed will also be applicable to understanding the role of co-transcriptional folding and assembly of ribozymes, regulatory RNA structures within non-coding and coding RNAs, and assembly of RNA-protein complexes such as the ribosome and spliceosome. These studies will also contribute understanding of the physical principles of out-of- equilibrium co-transcriptional RNA folding, help address long-standing questions about how dynamic RNA structures coordinate genetic processes, and shed light on how natural RNAs exploit out-of- equilibrium mechanisms to efficiently fold on extremely rugged free-energy landscapes. Since co-transcriptional RNA folding happens every time an RNA is synthesized, the broader impacts of this research include developing general principles and techniques that can be used to understand a wide array of fundamental cellular processes from gene expression to regulation. The study of riboswitches also has several broader impacts towards societal goals, since they can be used as biosensors within new molecular diagnostics, and they are important targets for new classes of antibiotics. Broader impacts of integrated research and education will come from a multi-pronged plan including conducting demonstrations of riboswitch diagnostics to school-age groups, mentorship of undergraduate researchers, and delivering hands-on tutorials of computational RNA folding approaches to broader scientific communities.The overarching goals of this proposal are to: (i) Uncover detailed mechanisms of how ligand binding bifurcates out-of-equilibrium RNA cotranscriptional folding pathways to enact genetic decisions in riboswitch RNAs; and (ii) Develop and apply new hybrid experimental-computational frameworks that can reconstruct RNA cotranscriptional folding pathways at the secondary and tertiary structure levels. The education plan focuses on integrating this research into hands-on demonstration activities targeted towards school age children, undergraduate researcher mentorship, and hands-on training tutorials for the broader scientific community. The post-genomic era has ushered in a new appreciation that RNAs play central roles in regulating, maintaining and defending the genomes of all organisms. However, a critical knowledge gap remains: we have relatively little understanding of the dynamic folding pathways that RNAs undergo as they are being synthesized during transcription, thus hindering our fundamental understanding of how RNA structures enact critical cellular functions such as catalysis, gene expression regulation, and cellular sensing. To address this gap, the PIs recently innovated and validated a hybrid experimental-computational approach that uses high-throughput RNA structure chemical probing data with computational algorithms to generate two and three-dimensional models of RNA cotranscriptional folding pathways. One central objective of this proposal is to extend this approach to incorporate more complex RNA structures and interactions such as pseudoknots relevant to a broad range of functional cellular RNAs. The second is to uncover biophysical principles of how out-of-equilibrium RNA fluctuations during cotranscriptional folding influence RNA function. The latter will be pursued through the use of riboswitch RNAs as model systems, which make ligand-mediated genetic decisions, use the dynamic formation of broadly utilized RNA structures to do so, and have broader impact relevance for fundamental biology and biotechnologies.This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Molecular Biophysics program in the Division of Molecular and Cellular Biosciences.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.

这项工作将揭示核糖开关 RNA 利用非平衡 RNA 折叠和配体结合来做出遗传决定的新原理。开发的工具还将适用于理解核酶的共转录折叠和组装、非编码和编码 RNA 内的调节 RNA 结构以及 RNA-蛋白质复合物（如核糖体和剪接体）的组装的作用。这些研究还将有助于理解不平衡共转录 RNA 折叠的物理原理，帮助解决有关动态 RNA 结构如何协调遗传过程的长期存在的问题，并阐明天然 RNA 如何利用不平衡现象。在极其崎岖的自由能地形上有效折叠的机制。由于每次合成 RNA 时都会发生共转录 RNA 折叠，因此这项研究的更广泛影响包括开发可用于理解从基因表达到调控的各种基本细胞过程的一般原理和技术。核糖开关的研究还对社会目标产生了一些更广泛的影响，因为它们可以用作新分子诊断中的生物传感器，并且它们是新型抗生素的重要目标。综合研究和教育的更广泛影响将来自多管齐下的计划，包括向学龄群体进行核糖开关诊断演示、对本科生研究人员进行指导，以及向更广泛的科学界提供计算 RNA 折叠方法的实践教程。该提案的目标是： (i) 揭示配体结合如何使不平衡的 RNA 共转录折叠途径分叉以在核糖开关 RNA 中制定遗传决定的详细机制； (ii) 开发和应用新的混合实验计算框架，可以在二级和三级结构水平上重建 RNA 共转录折叠途径。该教育计划的重点是将这项研究融入针对学龄儿童的实践示范活动、本科生研究人员指导以及针对更广泛的科学界的实践培训教程。后基因组时代带来了新的认识，即 RNA 在调节、维持和保护所有生物体的基因组方面发挥着核心作用。然而，仍然存在一个关键的知识差距：我们对 RNA 在转录过程中合成时所经历的动态折叠途径知之甚少，从而阻碍了我们对 RNA 结构如何发挥关键细胞功能（如催化、基因表达调控、和细胞传感。为了解决这一差距，PI 最近创新并验证了一种混合实验计算方法，该方法使用高通量 RNA 结构化学探测数据和计算算法来生成 RNA 共转录折叠途径的二维和三维模型。该提案的一个核心目标是扩展这种方法，以纳入更复杂的 RNA 结构和相互作用，例如与广泛的功能性细胞 RNA 相关的假结。第二个目标是揭示共转录折叠过程中不平衡的 RNA 波动如何影响 RNA 功能的生物物理学原理。后者将通过使用核糖开关 RNA 作为模型系统来实现，该系统做出配体介导的遗传决策，利用广泛使用的 RNA 结构的动态形成来实现这一点，并对基础生物学和生物技术产生更广泛的影响。该奖项由物理学部的生命系统物理学项目和分子与细胞生物科学部的分子生物物理学项目共同支持。该奖项反映了 NSF 的法定使命，并通过评估认为值得支持基金会的智力价值和更广泛的影响审查标准。