The RNA nanomachines of the gene expression machinery dissected at the single molecule level

在单分子水平上剖析基因表达机器的RNA纳米机器

• 批准号: 9920170
• 负责人: NILS G WALTER
• 金额: $ 84.78万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920170
• 关键词: Address; Bacterial RNA; Biochemical; Biological; Biology; Biophysics; Catalytic RNA; Cell physiology; Complex; Computer Simulation; Consensus; Coupling; Cryoelectron Microscopy; DNA-Directed RNA Polymerase; Dyes; Electron Microscopy; Electrostatics; Encapsulated; Enzymatic Biochemistry; Fluorescence Microscopy; Fluorescent Probes; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Individual; Kinetics; Length; Life; Ligands; Light; Messenger RNA; Outcome; Plant Roots; Process; RNA; RNA Folding; RNA Splicing; RNA analysis; RNA chemical synthesis; Research; Site; Spliceosomes; Structure; Structure-Activity Relationship; Thermodynamics; Thinking; Time; Transcript; Transcriptional Regulation; Translations; Untranslated RNA; base; gene function; molecular dynamics; nanomachine; nanoscale; single molecule; single-molecule FRET

TITLE: The RNA nanomachines of gene expression dissected at the single molecule level ABSTRACT: Over two decades, the Walter lab has contributed to the RNA field by building a broad research portfolio focused on dissecting the mechanisms of the nanoscale RNA machines of gene expression – ranging from small viroidal ribozymes and bacterial riboswitches to the eukaryotic spliceosome – by single molecule fluorescence microscopy. Leveraging this expertise, the two long-term goals of the current proposal are to: 1.) Apply our established mechanistic enzymology approaches to an ever broader set of RNAs involved in regulating transcription, translation and splicing, seizing the opportunities arising from the continuing discoveries of new functional RNAs. 2.) Push the limits of our approaches to be able to probe increasingly complex biological contexts and mechanisms since unexpected discoveries – as we found – often await where individual RNA nanomachines interact. In pursuit of these goals, we will address the overarching hypothesis that dynamic RNA structures are a major determinant of the outcomes of gene expression, often in ways that have been overlooked by a field that historically was rooted in genetics, where genes regularly were drawn as rectangular boxes, and function commonly was thought of as dictated by sequence rather than structure. Such thinking is countered by, for example, the fact that nascent RNA structure has a significant impact on transcription in the form of regulatory riboswitches embedded near the 5' ends of bacterial mRNAs and of transcription terminator hairpins at the 3' end. Conversely, the time-ordered, 5'-to-3' directional RNA synthesis of transcription often yields kinetically trapped RNA folds distinct from the most thermodynamically stable structure of a refolded full-length transcript. Encapsulating the power of our pursuit, we recently combined single-molecule, biochemical and computational simulation approaches to show that transcriptional pausing at a site immediately downstream of a riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced consensus pause sequence, and electrostatic and steric interactions with the exit channel of bacterial RNA polymerase. We posit that many more examples of such intimate structural and kinetic coupling between RNA folding and gene expression remain to be discovered, leading to the exquisite regulatory control and kinetic proofreading enabling all life processes. To reveal more such couplings, we will probe the dynamics of carefully purified transcriptional and translational riboswitch-containing, as well as spliceosomal, gene expression complexes using a tailored combination of single molecule fluorescence resonance energy transfer (smFRET), Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) based on super-resolved co-localization of RNA targets and fluorescent probes, cryo-electron microscopy – augmented by a proposed dye-based single molecule correlative light electron microscopy (smCLEM) – and, where appropriate, molecular dynamics simulations. We anticipate that these studies have the potential to transform our understanding of RNA structure-function relationships in general, and of how RNA structure is governing the function of cellular gene expression machines in particular.

标题： 在单分子水平上剖析基因表达的RNA纳米机器 抽象的： 二十年来，Walter 实验室通过建立广泛的研究组合，为 RNA 领域做出了贡献 剖析基因表达的纳米级RNA机器的机制——从小型病毒 核酶和细菌核糖开关连接真核剪接体——通过单分子荧光 利用这种专业知识，当前提案的两个长期目标是： 1.) 应用我们的技术。 建立了机械酶学方法来研究涉及调节的更广泛的 RNA 转录、翻译和剪接，抓住不断发现新事物所带来的机会 2.) 突破我们方法的极限，能够探测日益复杂的生物。 正如我们所发现的，意外的发现常常等待着个体 RNA 为了实现这些目标，我们将解决动态 RNA 的总体假设。 结构是基因表达结果的主要决定因素，但其方式往往被忽视 历史上植根于遗传学的领域，其中基因通常被绘制为矩形框，并且 功能通常被认为是由顺序而不是结构决定的，这种想法与以下观点相反。 例如，新生 RNA 结构以调控形式对转录产生重大影响。 核糖开关嵌入细菌 mRNA 的 5' 末端和 3' 转录终止子发夹附近 离线时，转录的按时间顺序的 5' 至 3' 定向 RNA 合成通常会产生动力学结果。 被捕获的 RNA 折叠与重折叠全长转录物的热力学最稳定的结构不同。 为了体现我们追求的力量，我们最近将单分子、生化和计算结合起来 模拟方法表明转录暂停在紧邻核糖开关下游的位点 需要新生 RNA 中存在无配体的假结、精确间隔的共有暂停序列，以及 我们假设还有更多与细菌 RNA 聚合酶出口通道的静电和空间相互作用。 RNA 折叠和基因表达之间这种密切的结构和动力学耦合的例子仍然存在 被发现，导致精确的调控和动力学校对，使所有生命过程得以实现。 揭示更多这样的耦合，我们将探讨仔细纯化的转录和翻译的动力学 含有核糖开关以及剪接体的基因表达复合物，使用定制的组合 单分子荧光共振能量转移 (smFRET)、RNA 的单分子动力学分析 基于 RNA 靶标和荧光的超分辨共定位的瞬时结构 (SiM-KARTS) 探针、冷冻电子显微镜——由提议的基于染料的单分子相关光增强 电子显微镜 (smCLEM) – 我们预计，在适当的情况下，还可以进行分子动力学模拟。 这些研究有可能改变我们对 RNA 结构与功能关系的理解 概述，以及 RNA 结构如何控制细胞基因表达机器的功能。