Studies of Riboswitch-Mediated Transcriptional Control Using Single-Molecule Fiel

利用单分子场进行核糖开关介导的转录控制的研究

• 批准号: 8695928
• 负责人: Ruben L Gonzalez
• 金额: $ 37.78万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-04 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8695928
• 关键词: 5' Untranslated Regions; Adenine; Antibiotics; Atomic Force Microscopy; Bacillus subtilis; Bacteria; Bacterial Genes; Binding; Biochemical; Biochemical Reaction; Biological Models; Carbon Nanotubes; Communicable Diseases; Complex; DNA Binding; DNA Sequence Rearrangement; DNA-Directed RNA Polymerase; Development; Devices; Diagnosis; Drug Targeting; Elements; Engineering; Enzymes; Escherichia coli; Event; Fluorescence Microscopy; Gene Expression; Genes; Genetic; Genetic Transcription; Hereditary Disease; Holoenzymes; In Vitro; Investigation; Kinetics; Knowledge; Label; Length; Mediating; Messenger RNA; Methods; Motion; Organism; Process; Property; Protein Complex Subunit; RNA; RNA Splicing; Resolution; Series; Specificity; Staging; Structure; Synthetic Genes; System; Thermodynamics; Time; Transcriptional Regulation; Transistors; Translations; aptamer; base; biological systems; biophysical techniques; design; fluorophore; improved; new technology; next generation; particle; pathogen; prevent; public health relevance; research study; response; single molecule; synthetic biology; tool; transcription termination

DESCRIPTION (provided by applicant): Riboswitches are genetic control elements located within the 5' untranslated regions of messenger RNAs (mRNAs) that undergo metabolite-dependent structural rearrangements to regulate mRNA transcription, splicing, translation, or stability in response to the presence and concentration of specific metabolites. The ubiquity of riboswitch-mediated transcriptional control in bacteria and the specificity with which riboswitches control bacterial gene expression are fueling efforts to develop next-generation antibiotics that target bacterial riboswitches. In addition, riboswitches are quickly becoming powerful tools in the field of synthetic biology, where they can be engineered to artificially control gene expression. Fully exploiting riboswitches for these applications, however, requires a detailed understanding of the mechanism of riboswitch-mediated transcriptional control. Although single-molecule (sm) biophysical methods, including sm fluorescence microscopy and sm force microscopy, have established themselves as powerful tools for studying metabolite- dependent structural rearrangements of riboswitches and transcription by RNA polymerases (RNAPs), the mechanistic information available from these sm methods remains limited by technical obstacles such as: (i) difficulties in fluorophore labeling of biomolecules; (ii) the application of invasive artificial forces; (iii) limited time resolution; and (iv) limited total observation time. The higly multi-disciplinary effort described here will expand upon recent development of a carbon nanotube-based sm field effect transistor (smFET) as a new, label-free, non-invasive, high-time-resolution, extended-observation-time, sm method for in vitro studies of biomolecular binding kinetics and structural dynamics. This smFET-based experimental system will be further developed to overcome many of the limitations of established sm methods, enabling the Bacillus subtilis pbuE adenine-responsive riboswitch and the corresponding B. subtilis RNAP to be used as a model system for studying the mechanisms of metabolite- dependent riboswitch structural rearrangement (Aim 1), transcription (Aim 2), and real-time riboswitch- mediated transcriptional control (Aim 3) at unprecedented time resolutions and throughputs. These studies will enable characterization of some of the most poorly defined aspects of the mechanism of riboswitch-mediated transcriptional regulation and will provide the tools and knowledge necessary to drive the development of new antibiotic drugs that target bacterial riboswitches and the design of new riboswitches that can be used to regulate synthetic gene networks.

描述（由申请人提供）：核糖开关是位于信使 RNA (mRNA) 5' 非翻译区域内的遗传控制元件，可根据代谢物的存在和浓度进行依赖于代谢物的结构重排，以调节 mRNA 转录、剪接、翻译或稳定性特定代谢物。细菌中核糖开关介导的转录控制的普遍性以及核糖开关的特异性 控制细菌基因表达正在推动开发针对细菌核糖开关的下一代抗生素。此外，核糖开关正迅速成为强大的工具 合成生物学领域，它们可以被设计来人工控制基因表达。然而，充分利用核糖开关进行这些应用需要详细了解核糖开关介导的转录控制机制。尽管单分子 (sm) 生物物理方法，包括 sm 荧光显微镜和 sm 力显微镜，已成为研究核糖开关代谢依赖性结构重排和 RNA 聚合酶 (RNAP) 转录的强大工具，但从这些 sm 中获得的机制信息方法仍然受到技术障碍的限制，例如：（i）生物分子荧光团标记的困难； (ii) 侵入性的应用 人工力量； (iii) 限时解决； (iv) 有限的总观察时间。 这里描述的高度多学科的努力将扩展基于碳纳米管的 sm 场效应晶体管 (smFET) 的最新发展，作为一种新型、无标记、非侵入性、高时间分辨率、延长观察时间、 sm 方法用于生物分子结合动力学和结构动力学的体外研究。这种基于 smFET 的实验系统将得到进一步开发，以克服现有 sm 方法的许多局限性，使枯草芽孢杆菌 pbuE 腺嘌呤响应核糖开关和相应的枯草芽孢杆菌 RNAP 能够用作研究代谢物机制的模型系统- 前所未有的依赖性核糖开关结构重排（目标 1）、转录（目标 2）和实时核糖开关介导的转录控制（目标 3）时间分辨率和吞吐量。这些研究将能够表征核糖开关介导的转录调控机制中一些最不明确的方面，并将提供必要的工具和知识，以推动针对细菌核糖开关的新抗生素药物的开发以及新核糖开关的设计用于调节合成基因网络。