Structure and Mechanism of SAM-responsive Riboswitches

SAM响应核糖开关的结构和机制

基本信息

批准号：
8516526
负责人：
Robert T Batey
金额：
$ 29.03万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
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/8516526
关键词：
5&apos 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

项目摘要

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.

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 响应核糖开关的结构多样性范围是多少，(2) SAM-I 未结合结构的性质是什么超家族核糖开关，（3）适体和表达域的哪些结构特征在调节中发挥功能作用，（4）结合热力学或动力学决定调节反应吗？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。。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。