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节手段是一种非蛋白质编码RNA元素，称为核糖开关。这些是在mRNA的领导者序列中发现的顺式作用元件，并通过将小分子代谢物与高度结构的受体结构域结合来调节基因表达。该受体将二级结构开关折叠在下游调节域中，后者又与表达机械（RNA聚合酶或核糖体）互动。在广泛的细菌，尤其是富公司和梭形细菌中，包括嘌呤，氨基酸和辅因子的生物合成和转运在内的中央代谢途径受核糖开关调节。此外，在许多医学重要的病原体中，包括单核细胞增生李斯特菌，金黄色葡萄球菌，铜绿假单胞菌和结核菌病的多种医学病原体中所必需的基因受到核糖开关的控制。此外，核糖开关越来越多地充当强大的模型系统，用于开发针对其他医疗兴趣RNA的小分子设计工具和方法。 为了建立对RNA如何与小分子相互作用的分子理解及其用于调节基因表达的机制的长期目标，我们使用s-腺苷甲硫代氨酸（SAM）结合核糖开关作为模型系统。该提案详细介绍了一组相互联系的特定目的，该目标解决了与这些研究目标相关的基本问题：（1）跨SAM响应性核糖开关的结构多样性的范围是多少，（2）SAM-I超级核心开关的未结构结构的性质是什么，（3）播放和表达型的结构范围内的构造和表达型的结构范围是构成的，该效果是构成的，该效果是构成的，该效果是构成的，（4）构成了型号（4个典型范围）（构成型号（4）。动力学决定了调节反应？为了解决这些问题，在一组专门设计用于研究结构/功能链接的实验中，将使用包括X射线晶体学，小角度晶体学，小角度X射线散射（SAX）以及各种生化和分子生物学方法的组合。对RNA如何特异性与小分子相互作用的更深入的了解将有助于为新一代的疗法奠定靶向非蛋白质编码RNA的新一代疗法，这些RNA在细菌和真核生物中都普遍存在。 公共卫生相关性：核糖开关是细菌中广泛使用的基于RNA的基因调节的一种形式，包括许多具有医学重要的病原菌，例如单核细胞增生李斯特菌，金黄色葡萄球菌，结核分枝杆菌，P。p。eruginosa和p。eruginosa和链球菌。拟议的研究试图通过直接结合小分子来调节细菌中这些RNA如何调节基因表达的原子水平了解。这些研究为我们如何通过基于结构的药物设计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来进一步了解如何利用RNA作为治疗剂的靶标。