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

标题： 在单分子水平上剖析基因表达的RNA纳米机器 摘要： 二十多年来，沃尔特实验室通过建立广泛的研究组合为rna领域做出了贡献。 关于基因表达的纳米级RNA机器的机制--从小类病毒开始 核酶和细菌核糖开关与真核剪接小体的单分子荧光法 显微镜。利用这一专门知识，当前提案的两个长期目标是：1.应用我们的 建立了机制酶学方法，以获得参与调控的更广泛的RNA集合 转录、翻译和拼接，抓住不断发现的新事物带来的机遇 功能核糖核酸。2.)突破我们方法的极限，能够探索日益复杂的生物学 背景和机制，因为意外的发现--正如我们所发现的--通常等待着单个RNA 纳米机器相互作用。在追求这些目标的过程中，我们将解决动态RNA 结构是基因表达结果的主要决定因素，通常以被忽视的方式 通过一个历史上植根于遗传学的领域，基因经常被绘制成矩形方框，以及 功能通常被认为是由顺序而不是结构决定的。这样的想法被反驳为， 例如，新生的RNA结构以调节的形式对转录有显著的影响 细菌mRNA5‘端附近嵌入的核糖开关和3’端转录终止子发夹 结束。相反，转录的5‘到3’定向RNA合成往往以动力学的方式产生 捕获的RNA折叠不同于重折叠的全长转录本的热力学最稳定的结构。 概括了我们追求的力量，我们最近将单分子、生化和计算结合在一起 模拟方法显示转录暂停在紧靠核糖开关下游的位置 在新生的RNA中需要一个无配体的假结，一个精确间隔的共识暂停序列，以及 静电和空间相互作用与细菌RNA聚合酶的出口通道。我们假设更多的人 RNA折叠和基因表达之间如此密切的结构和动力学耦合的例子仍然存在 被发现，导致精致的监管控制和动态校对，使所有生命过程。至 揭示更多这样的耦合，我们将探索仔细纯化的转录和翻译的动力学 含有核糖开关的基因表达复合体以及剪接体基因表达复合体 单分子荧光共振能量转移(SmFRET)、RNA单分子动力学分析 基于RNA靶标和荧光超分辨共定位的瞬时结构(SIM-KARTS) 低温电子显微镜探针，由建议的染料基单分子相关光增强 电子显微镜(SmCLEM)-以及适当的分子动力学模拟。我们预料到 这些研究有可能改变我们对RNA结构-功能关系的理解 一般，特别是RNA结构如何支配细胞基因表达机器的功能。

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.