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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。

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）

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.
影响因子：
0
作者：
Tinsley,RebeccaA;Harris,DinariA;Walter,NilsG
通讯作者：
Walter,NilsG

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.

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
影响因子：
0
作者：
Johnson-Buck AE;McDowell SE;Walter NG
通讯作者：
Walter NG

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.

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。