Structure and Mechanism of SAM-responsive Riboswitches

SAM响应核糖开关的结构和机制

• 批准号: 8369542
• 负责人: Robert T Batey
• 金额: $ 30.11万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8369542
• 关键词: 5' Untranslated Regions; Address; Alternative Splicing; Amino Acids; Anabolism; Attenuated; Bacteria; Bacterial Physiology; Binding; Biochemical; Biological; Biological Assay; Biological Models; Biology; Cartoons; Cell physiology; Chemicals; Code; Complement; Complex; Coupling; DNA-Directed RNA Polymerase; Data; Defect; Development; Discrimination; Drug Design; Elements; Environment; Eukaryota; Family; Figs - dietary; Foundations; Fusobacteria; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Transcription; Goals; Health; Homocysteine; Homocystine; Human Microbiome; In Vitro; Kinetics; Knowledge; Lead; Ligand Binding; Ligands; Link; Listeria monocytogenes; Malignant Neoplasms; Maps; Mediating; Medical; Medicine; Messenger RNA; Metabolic Pathway; Methodology; Modeling; Molecular; Mycobacterium tuberculosis; Nature; Outcome; Play; Process; Property; Pseudomonas aeruginosa; Purines; RNA; Regulation; Research; Ribosomes; Roentgen Rays; Role; S-Adenosylmethionine; Site-Directed Mutagenesis; Staphylococcus aureus; Stream; Streptococcus; Structure; Subarachnoid Hemorrhage; Testing; Therapeutic; Thermodynamics; Time; Transcriptional Regulation; Translating; Variant; Virulence; X-Ray Crystallography; antimicrobial; aptamer; attenuation; base; cis acting element; cofactor; design; human disease; in vivo; insight; interest; member; novel; pathogen; pathogenic bacteria; purine; receptor; research study; response; rho; small molecule; therapeutic target; tool

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. PUBLIC HEALTH RELEVANCE: Riboswitches are a form of RNA-based gene regulation widely utilized in bacteria, including a number of medically important pathogenic bacteria such as L. monocytogenes, S. aureus, M. tuberculosis, P. aeruginosa and Streptococcus species. The proposed research seeks to develop an atomic-level understanding of how these RNAs regulate gene expression in bacteria through their ability to directly bind small molecules. These studies serve to further our understanding as to how to exploit RNAs as targets of therapeutics via structure-based drug design or manipulate bacterial physiology to address health issues related to the composition of human microbiome.

描述（由申请人提供）：细菌中广泛使用的遗传调控手段是称为核糖开关的非蛋白质编码RNA元件。这些是在 mRNA 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化的受体结构域来调节基因表达。该受体指导下游调节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）相互作用。在多种细菌中，特别是厚壁菌门和梭杆菌中，包括嘌呤、氨基酸和辅因子生物合成和运输在内的中心代谢途径均由核糖开关调节。此外，在许多医学上重要的病原体中，生存或毒力所必需的基因受到核糖开关的控制，包括单核细胞增多性李斯特菌、金黄色葡萄球菌、铜绿假单胞菌和结核分枝杆菌，这使得它们作为设计抗菌治疗的新靶标受到极大的关注。此外，核糖开关日益成为强大的模型系统，用于开发设计针对其他医学感兴趣的 RNA 的小分子的工具和方法。 为了实现对 RNA 如何与小分子相互作用及其调节基因表达的机制进行分子理解的长期目标，我们使用 S-腺苷甲硫氨酸 (SAM) 结合核糖开关作为模型系统。该提案详细介绍了一系列相互关联的具体目标，解决了与这些研究目标相关的基本问题：(1) SAM 响应性核糖开关的结构多样性范围是多少，(2) SAM-I 超家族核糖开关的未结合结构的性质是什么，(3) 适体和表达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 动力学决定监管反应？为了解决这些问题，将在一组专门设计用于研究结构/功能联系的实验中结合使用 X 射线晶体学、小角 X 射线散射 (SAXS) 以及各种生化和分子生物学方法等方法。更深入地了解 RNA 如何与小分子特异性相互作用，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。 公共健康相关性：核糖开关是一种基于 RNA 的基因调控形式，广泛应用于细菌中，包括许多医学上重要的致病菌，如单核细胞增生李斯特菌、金黄色葡萄球菌、结核分枝杆菌、铜绿假单胞菌和链球菌属。拟议的研究旨在从原子水平上理解这些 RNA 如何通过直接结合小分子的能力来调节细菌中的基因表达。这些研究有助于进一步了解如何通过基于结构的药物设计来利用 RNA 作为治疗靶标，或操纵细菌生理学来解决与人类微生物组组成相关的健康问题。