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'未翻译区域内的遗传控制元件，该区域经历了代谢物依赖性的结构重排，以调节对特定代谢物的响应和浓度的响应和浓度，以调节mRNA转录，剪接，翻译或稳定性。核糖开关介导的细菌转录控制的无处不在以及核糖开关的特异性 对照细菌基因表达正在助长靶向细菌核糖开关的下一代抗生素的努力。此外，核糖开关正在迅速成为强大的工具 合成生物学领域，可以在其中设计以人为地控制基因表达。但是，完全利用这些应用的核糖开关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）已确立了自己的强大工具，用于研究核糖开关的代谢物 - 依赖性的结构重排和通过RNA聚合酶（RNAPS）转录的转录（RNAPS）（RNAPS）（RNAPS），从这些SM方法中获得的机械信息仍然受到这些障碍的限制，例如，诸如Inflyec的障碍很难：（i）。 （ii）侵入性的应用 人造力量； （iii）有限的时间分辨率； （iv）有限的总观察时间。 此处描述的热情的多学科工作将扩大基于碳纳米管的SM场效应晶体管（SMFET）作为一种新的，无标签的，无标签的，无创的，高时间的，扩展的，扩展的观察时间，用于体外研究生物分子结合Kinetics和结构性动力学的方法。将进一步开发基于SMFET的实验系统，以克服已建立的SM方法的许多局限性，从而实现了枯草芽孢杆菌 - 可腺嘌呤反应性核糖开关和相应的枯草芽孢杆菌RNAP，可用作模型系统，以研究用于研究依赖性的结构及其现实（AIM）的机制（目标2）在前所未有的时间分辨率和吞吐量上的转录控制（AIM 3）。这些研究将使核糖开关介导的转录调控机制的某些定义最明确的方面表征，并将提供必要的工具和知识，以驱动针对细菌核糖开关的新抗生素药物的开发以及可用于调节合成基因网络的新核糖开关设计。