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

交钥匙荧光显微镜的管理补充：在单分子水平上揭示核糖开关机制

基本信息

批准号：
9894327
负责人：
NILS G WALTER
金额：
$ 24.98万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9894327
关键词：
5&apos Untranslated Regions 7-deazaguanine Address Administrative Supplement Adopted Affect Award Bacteria Bacterial RNA Behavior Binding Biochemical Biological Biological Assay Biological Models Biology Biophysics Buffers Calorimetry Catalysis Cell Extracts Cell physiology Code Communication Complex Computer Simulation Consensus Coupling Crowding Crystallization DNA-Directed RNA Polymerase Data Electrostatics Environment Enzymatic Biochemistry Escherichia coli Fluorescence Microscopy Funding Gene Expression Genes Genetic Transcription Goals Grant Guanine Home environment In Vitro Individual Infrastructure Isomerism Kinetics Label Learning Length Life Ligand Binding Ligands Mediating Messenger RNA Microscope Microscopy Molecular Molecular Biology Molecular Conformation National Institute of General Medical Sciences Outcome Pathway interactions Physiological Play Postdoctoral Fellow Process Protein Subunits RNA RNA Conformation RNA Folding RNA Sequences RNA Splicing RNA analysis Readiness Ribosomes Role Rotation Site Small RNA Speed Structure Students Techniques Temperature Testing Time Titrations Training Transcriptional Regulation Translation Initiation Translations Untranslated RNA Update Work X-Ray Crystallography aptamer biophysical analysis cofactor design fluorescence microscope gene function graduate student hairpin ribozyme innovation insight instrument instrumentation member nanomachine nanoscale novel parent grant response single molecule single-molecule FRET tool translation assay undergraduate student

项目摘要

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.

项目概要：该补充品将严格更新可用于母基金 Riboswitch 各个方面的工具在单分子水平上阐明的机制，以及目前 PI 的所有 3.5 NIGMS R01 资助待转换为单一 R35 MIRA 奖，题为“基因表达的 RNA 纳米机器” 在单分子水平上剖析了所提出的仪器 ONI 最关键的方面。 Nanoimager S 的多功能性、交钥匙就绪性和易用性将极大地促进。 PI 中不同的博士后研究员、研究生和本科生群体可以访问大量的单分子荧光显微镜工具反过来又被直接利用。由家长资助，重点是剖析基因纳米级RNA机器的机制基于我们团队在该领域 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更广泛的参与调节的 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。背景和机制，因为意想不到的发现经常等待着个体 RNA 纳米机器为了实现这些目标，我们将解决动态 RNA 结构是一个统一的假设。基因表达结果的主要决定因素，例如新生 RNA 结构以嵌入的调节核糖开关的形式对转录和翻译产生重大影响靠近细菌 mRNA 的 5' 末端，体现了我们的科学方法解决我们的问题的力量。假设，我们最近结合了单分子、生化和计算模拟工具来证明核糖开关下游位点的转录暂停需要无配体的假结新生 RNA、精确间隔的共有暂停序列以及静电和空间相互作用我们假设还有更多类似紧密结构的例子。 RNA折叠和基因表达之间的动力学耦合仍有待发现，从而导致了精细的研究为了揭示更多这样的耦合，我们将探讨其动态。使用单分子荧光共振的定制组合产生额外的基因表达复合物能量转移 (smFRET) 和 RNA 瞬时结构的单分子动力学分析 (SiM-KARTS)。到目前为止，这些追求的主要瓶颈是与我们的两个自制的相关的陡峭的学习曲线显微镜阻止新小组成员做出重大贡献，直到他们完成 1- 我们预计，ONI Nanoimager S 添加到我们的显微镜武器库中将会带来 2 年的培训。通过引入一种易于使用的工具来加快我们的进步速度，该工具可以帮助刚开始博士后、研究生和本科生可以快速独立使用，直至“毕业”到自制显微镜。

项目成果

期刊论文数量（75）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme.

分子动力学表明腺嘌呤亚氨基在发夹核酶的酸碱催化中具有多功能性。

DOI：
10.1261/rna.1416709
发表时间：
2009-04-01
期刊：
RNA
影响因子：
4.5
作者：
通讯作者：

Significant kinetic solvent isotope effects in folding of the catalytic RNA from the hepatitis delta virus.

动力学溶剂同位素对 δ 型肝炎病毒催化 RNA 的折叠有显着影响。

DOI：
发表时间：
2003-11-19
期刊：
影响因子：
15
作者：
Tinsley, Rebecca A;Harris, Dinari A;Walter, Nils G
通讯作者：
Walter, Nils G

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles.

高渗相分离：包含物、颗粒和细胞器之外的冷凝物。

DOI：
发表时间：
2021
期刊：
影响因子：
0
作者：
Jalihal, Ameya P;Schmidt, Andreas;Gao, Guoming;Little, Saffron R;Pitchiaya, Sethuramasundaram;Walter, Nils G
通讯作者：
Walter, Nils G

Precise tuning of bacterial translation initiation by non-equilibrium 5'-UTR unfolding observed in single mRNAs.

通过在单个 mRNA 中观察到的非平衡 5-UTR 解折叠来精确调节细菌翻译起始。