Administrative Supplement for a Turnkey Fluorescence Microscope: Riboswitch mechanism unraveled at the single molecule level

项目总结： 本附录将关键地更新可用于母基金核糖开关所有方面的仪器 在单分子水平上解开了机制，以及目前所有3.5 NIGMS R01授予的PI 正在等待转化为单一的R35 Mira奖，名为基因表达的RNA纳米机器 在单分子水平上进行解剖。建议的工具最关键的方面是ONI 纳米成像仪S以其多功能性、即用即用和易用性而闻名。这些功能将极大地促进 不同群体的博士后研究员、研究生和本科生在PI的访问 大量的单分子荧光显微镜工具。反过来，这些工具被直接利用 由父母资助，专注于剖析基因的纳米级RNA机器的机制 在单分子水平表达。基于我们集团在该领域长达20年的专业知识，我们的目标是：1. 将我们已建立的机械酶学方法应用于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，因为意想不到的发现通常等待着单个RNA纳米机器 互动。在追求这些目标的过程中，我们将解决动态RNA结构是一种 基因表达结果的主要决定因素，例如新生RNA结构 以嵌入的调节性核糖开关的形式对转录和翻译产生重大影响 在细菌mRNAs的5‘端附近。例证了我们科学方法的力量，以解决我们的 假设，我们最近结合了单分子、生化和计算模拟工具来表明 在紧邻核糖开关下游的位置转录暂停需要一个无配体的假结 新生的RNA，一个精确间隔的共识暂停序列，以及静电和空间相互作用 细菌RNA聚合酶的出口通道。我们假设，更多类似的亲密结构的例子 RNA折叠和基因表达之间的动力学耦合还有待发现，导致了精致的 启用所有生命过程的监管控制。为了揭示更多这样的耦合，我们将探索 使用定制的单分子荧光共振组合的附加基因表达复合体 能量转移(SmFRET)和RNA瞬时结构的单分子动力学分析(SIM-KARTS)。一个 到目前为止，这些追求的主要瓶颈是与我们的两个自制产品相关的陡峭学习曲线 显微镜，防止新的小组成员做出重大贡献，直到他们完成1- 经过2年的培训。我们预计，ONI纳米成像仪S加入我们的显微镜武器库将使 通过引入一种易于使用的工具，开始攻读博士后、研究生和 本科生可以快速独立使用，直到他们“毕业”到自制的显微镜。

PROJECT SUMMARY: This supplement will critically update the instrumentation available for all aspects of the parent grant, Riboswitch mechanism unraveled at the single molecule level, as well as all 3.5 NIGMS R01 grants of the PI that are currently pending conversion into a single R35 MIRA award, entitled The RNA nanomachines of gene expression dissected at the single molecule level. The most critical aspects of the proposed instrumentation, the ONI Nanoimager S, are its versatility, turnkey readiness, and ease of use. These features will dramatically facilitate access by the diverse group of postdoctoral fellows, graduate students and undergraduate students in the PI’s group to a plethora of single molecule fluorescence microscopy tools. In turn, these tools are leveraged directly by the parent grant, which is focused on dissecting the mechanisms of the nanoscale RNA machines of gene expression at the single molecule level. Building on our group’s 20-year expertise in this space, we aim 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 often await where individual RNA nanomachines interact. In pursuit of these aims, we will address the unifying hypothesis that dynamic RNA structures are a major determinant of the outcomes of gene expression, as exemplified by the fact that nascent RNA structure has a significant impact on both transcription and translation in the form of regulatory riboswitches embedded near the 5’ ends of bacterial mRNAs. Exemplifying the power of our scientific approach to address our hypothesis, we recently combined single-molecule, biochemical and computational simulation tools 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 similarly intimate structural and kinetic coupling between RNA folding and gene expression remain to be discovered, leading to the exquisite regulatory control enabling all life processes. To reveal more such couplings, we will probe the dynamics of additional gene expression complexes using a tailored combination of single molecule fluorescence resonance energy transfer (smFRET) and Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS). A major bottleneck in these pursuits so far has been the steep learning curve associated with our two home-built microscopes that keeps new group members from making significant contributions until they have completed 1- 2 years of training. We anticipate that addition of the ONI Nanoimager S to our microscopy arsenal will transform the speed of our progress by introducing an easy-to-use instrument that beginning postdocs, graduate and undergraduate students can quickly use independently until they “graduate” to the home-built microscopes.