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。 解剖基因表达的纳米RNA机器的机制-从小病毒 核酶和细菌核糖开关的真核剪接体-通过单分子荧光 显微镜利用这一专业知识，目前提案的两个长期目标是：1。适用我们 建立了一个机制酶学方法，以更广泛的RNA参与调节 转录，翻译和拼接，抓住新的发现带来的机会， 功能性RNA。2.）的情况。推动我们的方法的极限，以便能够探测日益复杂的生物学 背景和机制，因为出乎意料的发现-正如我们所发现的-经常等待个体RNA 纳米机器相互作用。在追求这些目标的过程中，我们将讨论动态RNA 结构是基因表达结果的一个主要决定因素，通常以被忽视的方式 这个领域在历史上植根于遗传学，基因通常被画成矩形框， 功能通常被认为是由序列而不是结构决定的。这种想法被反驳， 例如，新生RNA结构对转录具有显著的影响，其形式是调节转录， 核糖开关嵌入在细菌mRNA的5'端附近和转录终止子发夹的3'端附近。 端相反，转录的时间顺序，5 '到3'方向的RNA合成通常在动力学上产生 捕获的RNA折叠不同于重折叠的全长转录物的最稳定的结构。 为了封装我们追求的力量，我们最近将单分子、生物化学和计算 模拟方法显示转录暂停在核糖开关下游的一个位点 需要新生RNA中的无配体假结，精确间隔的共有暂停序列，以及 与细菌RNA聚合酶的出口通道的静电和空间相互作用。我们会再做更多 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，导致精致的监管控制和动力学校对，使所有生命过程。到 揭示更多这样的耦合，我们将探测仔细纯化的转录和翻译动力学 含有核糖开关以及剪接体的基因表达复合物， 单分子荧光共振能量转移（smFRET），RNA的单分子动力学分析 瞬时结构（SiM-KARTS）基于RNA靶标和荧光的超分辨共定位 探针，低温电子显微镜-由建议的基于染料的单分子相关光增强 电子显微镜（smCLEM）-和，在适当的情况下，分子动力学模拟。我们预计 这些研究有可能改变我们对RNA结构-功能关系的理解， 一般来说，以及RNA结构如何控制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.