In vitro and in vivo characterization of the cyclic-di-GMP riboswitch

环二 GMP 核糖开关的体外和体内表征

• 批准号: 9241885
• 负责人: Katherine D Launer-Felty
• 金额: $ 5.71万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9241885
• 关键词: 5' Untranslated Regions; Animal Model; Anthrax disease; Bacillus cereus; Bacteria; Bacterial Genes; Binding; Biochemistry; Biological Assay; Biological Models; Botulism; Broccoli - dietary; Cell Survival; Cell membrane; Cells; Characteristics; Charge; Cholera; Complex; Crystallization; Disease; Electrophoretic Mobility Shift Assay; Elements; Environment; Fluorescence; Gene Expression; Genes; Genetic; Gentian Violet; Gram-Negative Bacteria; High-Throughput Nucleotide Sequencing; Hydrolysis; In Vitro; Knock-out; Laboratories; Ligand Binding; Ligands; Messenger RNA; Methods; Microbial Biofilms; Microbiology; Molecular; Molecular Conformation; Monitor; Organism; Pathway interactions; Periodicity; Phenotype; Proteins; Protocols documentation; Publishing; RNA; Resistance; Role; Second Messenger Systems; Series; Signal Pathway; Signal Transduction; Staining method; Stains; Structure; System; Tertiary Protein Structure; Testing; Tetanus; Therapeutic; Untranslated RNA; Virulence; Virulence Factors; analog; aptamer; behavioral study; cell motility; diguanylate cyclase; experimental study; in vivo; interdisciplinary approach; phosphoric diester hydrolase; public health relevance; receptor; resazurin; response; scale up; small molecule; structural biology; uptake

 DESCRIPTION (provided by applicant): Riboswitches are structured RNAs that undergo significant conformational changes in response to specific ligands as a mechanism to regulate gene expression. In addition to several proteins, two different classes of c-di-GMP riboswitches have been implicated in the cyclic diguanosine monophosphate (c-di-GMP) second-messenger pathway, a ubiquitous signaling method in bacteria. The crystal structures for both classes of c-di-GMP riboswitches have been solved showing two different mechanisms for c-di-GMP recognition. These riboswitches are located in the 5'-untranslated regions of genes involved in biofilm formation, cell motility, and virulence factors. Since c-di-GMP riboswitches are prevalent in bacterial species responsible for tetanus, anthrax, botulism, and cholera among others, their characterization has direct therapeutic applications. Although the structure of both classes of riboswitches has been extensively characterized, very few experiments have been performed in vivo. The Strobel lab has synthesized a series of c-di-GMP analogues to differentially study the ligand binding characteristics in both c-di-GMP riboswitches. Several of these ligands also show resistance to intracellular cleavage and are ideal for studying the behavior of c-di-GMP riboswitches in the cell. This proposal seeks to determine the functions of c-di-GMP in the cell and define the role of the c-di-GMP riboswitch on bacterial gene expression using a multidisciplinary approach spanning biochemistry, structural biology, microbiology, and genetics.

 描述（由申请人提供）：核糖开关是结构化的RNA，其响应于作为调节基因表达的机制的特定配体而经历显著的构象变化。除了几种蛋白质外，两种不同类型的c-di-GMP核糖开关也参与了环二磷酸鸟苷（c-di-GMP）第二信使途径，这是细菌中普遍存在的信号传导方法。两类c-di-GMP核糖开关的晶体结构已被解析，显示出c-di-GMP识别的两种不同机制。这些核糖开关位于参与生物膜形成、细胞运动和毒力因子的基因的5 '-非翻译区。由于c-di-GMP核糖开关在引起破伤风、炭疽、肉毒杆菌中毒和霍乱等的细菌物种中普遍存在，因此它们的表征具有直接的治疗应用。虽然这两类核糖开关的结构已被广泛表征，但很少有实验在体内进行。Strobel实验室合成了一系列c-di-GMP类似物，以差异化研究两种c-di-GMP核糖开关中的配体结合特征。这些配体中的几种还显示出对细胞内裂解的抗性，并且是研究细胞中c-di-GMP核糖开关行为的理想选择。该提案旨在确定c-di-GMP在细胞中的功能，并使用跨生物化学，结构生物学，微生物学和遗传学的多学科方法来定义c-di-GMP核糖开关对细菌基因表达的作用。