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

• 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
• 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' 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

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： 这一补充将批判性地更新仪器可用于所有方面的家长赠款，核糖开关 在单分子水平上解开了机制，以及目前正在研究的PI的所有3.5 NIGMS R 01赠款 即将转换为单一的R35 MIRA奖，题为基因表达的RNA纳米机器 在单分子水平上解剖。建议的仪器，ONI的最关键的方面 Nanoimager S的最大优点是其多功能性、交钥匙准备和易用性。这些功能将极大地促进 访问由博士后研究员，研究生和本科生在PI的不同群体 使用大量单分子荧光显微镜工具。反过来，这些工具又被直接利用， 由父母资助，这是专注于解剖基因的纳米RNA机器的机制， 在单分子水平上表达。基于我们集团在该领域20年的专业知识，我们的目标是：1. 将我们建立的机制酶学方法应用于更广泛的RNA组，这些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，因为意想不到的发现往往等待个别RNA纳米机器 互动.在追求这些目标的过程中，我们将讨论一个统一的假设，即动态RNA结构是 基因表达结果的主要决定因素，如新生RNA结构 对转录和翻译都有重要的影响， 在细菌mRNA的5'末端附近。以科学方法的力量为例， 假设，我们最近结合单分子，生物化学和计算模拟工具，以表明， 在核糖开关下游的转录暂停需要一个无配体的假结， 新生RNA，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。我们发现，更多类似的亲密结构的例子， 以及RNA折叠和基因表达之间的动力学耦合仍有待发现，这导致了基因表达的精细化。 调控使所有生命过程。为了揭示更多这样的耦合，我们将探索 使用单分子荧光共振的定制组合的另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）。一 到目前为止，这些追求的主要瓶颈是与我们的两个自制的 显微镜，使新的小组成员作出重大贡献，直到他们已经完成1- 两年的训练。我们预计，ONI Nanoimager S加入我们的显微镜库将改变 我们的进展速度，通过引入一个易于使用的工具，开始博士后，毕业生和 本科生可以很快独立使用，直到他们“毕业”到自制的显微镜。

项目成果

Magnesium dependence of the amplified conformational switch in the trans-acting hepatitis delta virus ribozyme.

反式作用丁型肝炎病毒核酶中放大构象开关的镁依赖性。

• DOI: 10.1021/bi049471e
• 发表时间: 2004
• 期刊: Biochemistry.
• 作者: Tinsley,RebeccaA;Harris,DinariA;Walter,NilsG
• 通讯作者: Walter,NilsG

Metal ions: supporting actors in the playbook of small ribozymes.

金属离子：小核酶剧本中的辅助演员。

• DOI: 10.1039/9781849732512-00175
• 发表时间: 2011
• 期刊: Metal ions in life sciences
• 作者: Johnson-Buck AE;McDowell SE;Walter NG
• 通讯作者: Walter NG

The genomic HDV ribozyme utilizes a previously unnoticed U-turn motif to accomplish fast site-specific catalysis.

基因组HDV核酶利用先前未忽略的掉头基序来完成快速位点特异性催化。

• DOI: 10.1093/nar/gkl1104
• 发表时间: 2007
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Sefcikova, Jana;Krasovska, Maryna V.;Sponer, Jiri;Walter, Nils G.
• 通讯作者: Walter, Nils G.

RNA chaperones stimulate formation and yield of the U3 snoRNA-Pre-rRNA duplexes needed for eukaryotic ribosome biogenesis.

• DOI: 10.1016/j.jmb.2009.05.072
• 发表时间: 2009-07-31
• 期刊: JOURNAL OF MOLECULAR BIOLOGY
• 影响因子: 5.6
• 作者: Gerczei, Timea;Shah, Binal N.;Manzo, Anthony J.;Walter, Nils G.;Correll, Carl C.
• 通讯作者: Correll, Carl C.

Solution structure of an alternate conformation of helix27 from Escherichia coli16S rRNA.

• DOI: 10.1002/bip.21626
• 发表时间: 2011-10
• 期刊: BIOPOLYMERS
• 影响因子: 2.9
• 作者: Spano, Meredith Newby;Walter, Nils G.
• 通讯作者: Walter, Nils G.