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: 10613420
• 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/10613420
• 关键词: 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; 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; gene function; molecular dynamics; nanomachine; nanoscale; single molecule; single-molecule FRET; ultra high resolution

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的基因表达的RNA纳米机器 抽象的： 二十年来，沃尔特实验室通过建立以广泛的研究组合为重点为RNA领域做出了贡献 解剖基因表达的纳米级RNA机的机理 - 范围从小病毒 核酶和细菌核糖开关到真核剪接体 - 单分子荧光 显微镜。利用这一专业知识，当前建议的两个长期目标是：1。）应用我们 既定的机械酶学方法 转录，翻译和拼接，抓住了持续发现的新机会 功能性RNA。 2.）推动我们的方法的极限，以探测日益复杂的生物学 上下文和机制以来我们发现的意外发现，通常会等待单个RNA的位置 纳米机器相互作用。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确定者，通常以被忽视的方式 通过历史上植根于遗传学的领域，经常将基因作为矩形盒子绘制，并且 函数通常被认为是由序列而不是结构决定的。这样的思想是由 例如，新生的RNA结构对调节形式的转录具有重大影响 在细菌mRNA的5端和3'的转录终结器发夹附近嵌入的核糖开关 结尾。相反，时间订购的5'至3'定向RNA的转录合成通常会产生 捕获的RNA折叠与重折叠的全长转录本的最热力学稳定结构不同。 封装我们追求的力量，我们最近将单分子，生化和计算结合在一起 模拟方法以表明在核糖开关下游的站点上的转录暂停 需要在新生的RNA中使用无配体的伪not，一个精确的共识暂停序列，并且 静电和空间与细菌RNA聚合酶出口通道的相互作用。我们指出更多 RNA折叠和基因表达之间这种亲密的结构和动力学耦合的例子 被发现，导致独家的监管控制和动力学校对，从而实现所有生命过程。到 揭示更多此类耦合，我们将探测精心纯化的转录和翻译的动力学 使用量身定制的组合，含核糖开关的基因表达复合物以及剪接的基因表达复合物 单分子荧光共振能传递（SMFRET），RNA的单分子动力学分析 基于RNA靶标和荧光的超级共定位的瞬态结构（SIM卡丁车） 探针，冷冻电子显微镜 - 由提出的基于染料的单分子相关光增强 电子显微镜（SMCLEM） - 以及适当的分子动力学模拟。我们预料到这一点 这些研究有可能改变我们对RNA结构功能关系的理解 一般以及RNA结构如何管理细胞基因表达机的功能。