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

描述(申请人提供)：核糖开关是位于信使RNA(MRNAs)的5‘非翻译区内的遗传控制元件，它通过依赖代谢物的结构重排来调节mRNA的转录、剪接、翻译或稳定性，以响应特定代谢物的存在和浓度。核糖开关介导的转录调控在细菌中的普遍存在及其特异性 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，核糖开关正在迅速成为 在合成生物学领域，可以对它们进行工程改造，以人工控制基因表达。然而，要充分利用核糖开关进行这些应用，需要对核糖开关介导的转录控制机制有详细的了解。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，已经成为研究核糖开关的代谢物依赖的结构重排和rna聚合酶(Rna)转录的有力工具，但这些sm方法提供的机制信息仍然受到技术障碍的限制：(I)生物分子的荧光团标记困难；(Ii)侵入性的应用。 (3)有限的时间分辨率；(4)有限的总观测时间。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，作为一种新的、无标记、非侵入性、高时间分辨率、延长观察时间的sm方法，用于体外研究生物分子结合动力学和结构动力学。这个基于smFET的实验系统将被进一步开发，以克服现有sm方法的许多局限性，使枯草杆菌pbuE腺嘌呤响应型核糖开关和相应的枯草杆菌RNAP能够作为模型系统，以前所未有的时间分辨率和吞吐量研究代谢物依赖的核糖开关结构重排(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)的机制。这些研究将能够描述核糖开关介导的转录调控机制中一些最模糊的方面，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，以及可用于调节合成基因网络的新核糖开关的设计。

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.