Basis of Gene Regulation by Purine and Cobalamine Riboswitches

项目摘要。 细菌中广泛存在的一种基因调控机制是由一组非编码的RNA元件组成，称为 核糖开关。这些都是在mRNAs的前导序列中发现的顺式作用元件，并调节基因 通过它们将特定的效应分子直接结合到高结构适配子的能力来表达 域。与适配子结构域结合的效应器被传递到下游二级结构开关 在指示表达机制的表达平台中(通常是RNA聚合酶或 核糖体)。在广泛的细菌中，特别是菲尔米特和梭杆菌，中心代谢 包括嘌呤、氨基酸和辅因子的生物合成和运输途径都受核糖开关的调控。 此外，在许多医学领域，对生存或毒力至关重要的基因都处于核糖开关的控制之下。 重要病原体包括单核细胞增多性李斯特菌、金黄色葡萄球菌、铜绿假单胞菌、 艰难梭菌和结核分枝杆菌使它们成为新的靶标 抗菌疗法。同样重要的是，核糖开关是理解 RNA生物学的各个方面，包括结构、折叠和调节活性的机制以及 开发工具和方法来设计针对其他医学上有价值的RNA的小分子。 朝着发展对RNA如何与小分子相互作用的分子理解的长期目标 分子和它用来调节基因表达的机制，我们正在使用嘌呤和钴胺- 结合核糖开关作为模型系统。该提案详细说明了一系列相互关联的具体目标 解决与这些研究目标有关的基本问题：(1)作图顺序和结构 结构转换之外的表达平台特征对有效的配体依赖至关重要 监管活动，(2)了解如何调节高阶三级结构的结构性“模块” 有助于快速有效地折叠适体结构域，以及(3)研究适体的可塑性 通过它们识别不同配体的能力来识别结构域。为了解决这些问题，我们结合了以下几个方面 结构(x射线结晶学)、生物物理(量热法和停流动力学)、生化(化学 足迹)、遗传和分子生物学(体内和体外活性分析)和 生物信息学/计算方法将结合起来研究结构-调控活性之间的联系 核糖开关。值得注意的是，这项建议采用了“功能优先”的研究策略，而不是 “结构优先”方法主导了当前对核糖开关的研究，以建立更强的联系 RNA结构和功能之间的关系。更深入地了解RNA如何与Small相互作用 影响其结构和活性的分子将有助于正在进行的开发新一代 针对细菌和真核生物中的非蛋白质编码RNA的治疗学。

Project Summary. A widespread mechanism of gene regulation in bacteria is by a group of noncoding RNA elements called a riboswitch. These are cis-acting elements found in the leader sequence of mRNAs and regulate gene expression through their ability to directly bind a specific effector molecule to a highly-structured aptamer domain. Effector binding to the aptamer domain is communicated to a downstream secondary structural switch in the expression platform that instructs the expression machinery (generally RNA polymerase or the ribosome). In a broad spectrum of bacteria, particularly Firmicutes and Fusobacteria, central metabolic pathways including purine, amino acid, and cofactor biosynthesis and transport are regulated by riboswitches. Furthermore, genes essential for survival or virulence are under riboswitch control in numerous medically important pathogens including Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium difficile, and Mycobacterium tuberculosis making them of great interest as novel targets for antimicrobial therapeutics. Of equal importance, riboswitches are powerful model systems for understanding various aspects of RNA biology including structure, folding and mechanisms of regulatory activity along with developing tools and methodologies for designing small molecules that target other RNAs of medical interest. Towards the long-term goal of developing a molecular understanding of how RNA interacts with small molecules and the mechanisms it uses to regulate gene expression, we are using purine- and cobalamin- binding riboswitches as model systems. This proposal details a set of interconnected specific aims that addresses fundamental questions related to these research goals: (1) mapping sequence and structural features of the expression platform beyond the structural switch crucial for efficient ligand-dependent regulatory activity, (2) understanding how structural “modules” that mediate higher-order tertiary structure contribute to rapid and efficient folding of the aptamer domain, and (3) investigating the plasticity of aptamer domains through their ability to recognize different ligands. To address these questions, a combination of structural (x-ray crystallography) biophysical (calorimetry and stopped-flow kinetics), biochemical (chemical footprinting), genetic and molecular biological (in vivo and in vitro activity assays) and bioinformatics/computational approaches will be combined to study the structure-regulatory activity linkage in riboswitches. Significantly, this proposal adopts a “function-first” research strategy, as opposed to the “structure-first” approach that dominates current research into riboswitches to make a stronger connection between RNA structure and function. A deeper knowledge of how RNA specifically interacts with small molecules that affects its structure and activity will contribute to ongoing efforts to develop a new generation of therapeutics that target non-protein coding RNAs in bacteria and eukaryotes.