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

这项工作将揭示核糖开关RNA利用失衡的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中的调节RNA结构以及核糖体和剪接体等RNA-蛋白质复合体的组装。这些研究还将有助于理解非平衡共转录RNA折叠的物理原理，有助于解决关于动态RNA结构如何协调遗传过程的长期存在的问题，并阐明自然RNA如何利用非平衡机制在极其崎岖的自由能景观中高效折叠。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广泛基本细胞过程的一般原理和技术。对核糖开关的研究对社会目标也有几个更广泛的影响，因为它们可以在新的分子诊断中用作生物传感器，并且它们是新类别抗生素的重要靶标。综合研究和教育的更广泛影响将来自一个多管齐下的计划，包括向学龄组进行核糖开关诊断示范，指导本科生研究人员，并向更广泛的科学界提供计算RNA折叠方法的动手教程。这项提议的首要目标是：(I)揭示配体结合如何分叉失衡的RNA共转录折叠途径的详细机制，以制定核糖开关RNA中的遗传决定；以及(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重建RNA共转录折叠路径。教育计划的重点是将这项研究纳入针对学龄儿童、本科生研究人员指导和更广泛科学界的实践培训教程的实践示范活动。后基因组时代带来了一种新的认识，即RNA在调节、维持和保护所有生物的基因组方面发挥着核心作用。然而，一个关键的知识缺口仍然存在：我们对RNA在转录过程中合成时经历的动态折叠路径了解相对较少，从而阻碍了我们对RNA结构如何发挥关键细胞功能的基本理解，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共转录折叠路径的二维和三维模型。这项提议的一个中心目标是扩展这一方法，以纳入更复杂的RNA结构和相互作用，例如与广泛的功能细胞RNA相关的假结。第二个是揭示共转录折叠过程中失衡的RNA波动如何影响RNA功能的生物物理原理。后者将通过使用核糖开关RNA作为模型系统来进行，它做出配体介导的遗传决策，使用广泛使用的RNA结构的动态形成来这样做，并对基础生物学和生物技术具有更广泛的影响相关性。该项目由物理部的生命系统物理计划和分子和细胞生物科学部的分子生物物理学计划联合支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.