Structure and Mechanism of SAM-responsive Riboswitches

描述(申请人提供)：在细菌中广泛使用的遗传调控手段是一种称为核糖开关的非蛋白质编码的RNA元件。这些是在mRNAs的前导序列中发现的顺式作用元件，通过将小分子代谢物直接结合到高度结构的受体结构域来调节基因的表达。这种受体指导下游调控结构域中二级结构开关的折叠，进而与表达机制(RNA聚合酶或核糖体)接口。在广泛的细菌中，特别是Firmicuts和梭杆菌，中央代谢途径包括嘌呤、氨基酸和辅因子的生物合成和运输都受到核糖开关的调控。此外，在许多医学上重要的病原体中，如单核细胞增生性李斯特菌、金黄色葡萄球菌、铜绿假单胞菌和结核分枝杆菌，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，这使得它们成为设计抗菌药物的新靶点。此外，核糖开关越来越多地作为强大的模型系统，用于开发工具和方法来设计以其他医学感兴趣的RNA为靶点的小分子。为了从分子上了解核糖核酸如何与小分子相互作用及其调节基因表达的机制，我们使用了S-腺苷甲硫氨酸结合的核糖开关作为模型系统。这项建议详细说明了一系列相互关联的具体目标，以解决与这些研究目标相关的基本问题：(1)SAM反应核糖开关的结构多样性范围是什么，(2)SAM-I超家族核糖开关的非结合结构的性质是什么，(3)适体和表达结构域的哪些结构特征在调节中发挥功能作用，以及(4)结合热力学或动力学是否决定调节反应？为了解决这些问题，将利用X射线结晶学、小角X射线散射(SAXS)以及各种生化和分子生物学方法进行一系列专门设计的实验，以研究结构/功能联系。对RNA如何与小分子具体相互作用的更深入了解，将有助于为针对细菌和真核生物中普遍存在的非蛋白质编码RNA的新一代疗法铺平道路。

DESCRIPTION (provided by applicant): A widely used means of genetic regulation in bacteria is a non-protein coding RNA element called a riboswitch. These are cis-acting elements found in the leader sequence of mRNAs and regulate gene expression by directly binding small molecule metabolites to a highly structured receptor domain. This receptor directs folding of a secondary structural switch in a downstream regulatory domain that in turn interfaces with the expression machinery (either 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 a number of medically important pathogens including Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis making them of great interest as novel targets for designing antimicrobial therapeutics. In addition, riboswitches are increasingly serving as powerful model systems for developing the tools and methodologies for the design of 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 S-adenosylmethionine (SAM)-binding riboswitches as a model system. This proposal details a set of interconnected specific aims that addresses fundamental questions related to these research goals: (1) what is the range of structural diversity across SAM-responsive riboswitches, (2) what is the nature of the unbound structure of SAM-I superfamily riboswitches, (3) which structural features of the aptamer and expression domains play functional roles in regulation, and (4) do binding thermodynamics or kinetics dictate the regulatory response? To address these questions, a combination of approaches including X-ray crystallography, small-angle X-ray scattering (SAXS), and various biochemical and molecular biological approaches will be utilized in a set of experiments specifically designed to study the structure/function linkage. A deeper knowledge of how RNA specifically interacts with small molecules will help pave the way for a new generation of therapeutics that target non-protein coding RNAs that are pervasive in both bacteria and eukaryotes.