Resolving Structure and Mechanism of tRNA-actuated riboswitches

解析 tRNA 驱动核糖开关的结构和机制

• 批准号: 9107101
• 负责人: EDWARD P NIKONOWICZ
• 金额: $ 32.24万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9107101
• 关键词: Address; Affect; Affinity; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Anticodon; Apical; Attenuated; Bacillus anthracis; Base Sequence; Binding; Biological Assay; Boxing; Cells; Charge; Codon Nucleotides; Collaborations; Communication; Complex; Coupled; Coupling; Data; Defect; Dissociation; Elbow; Elements; Engineering; Ensure; Enzymes; Essential Genes; Event; Fluorescence Microscopy; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic Engineering; Genetic Transcription; Glycine-Specific tRNA; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Growth; In Situ; Kinetics; Knowledge; Ligand Binding; Ligands; Link; Manuscripts; Measurement; Mediating; Metabolic; Methods; Microbial Biofilms; Modeling; Modification; Molecular Conformation; Monitor; Nucleotides; Performance; Physiological; Physiology; Preparation; RNA; Regulation; Research Personnel; Roentgen Rays; Serine-Specific tRNA; Structure; System; Techniques; Temperature; Testing; Time; Tissue-Specific Gene Expression; Transcript; Transcriptional Regulation; Transfer RNA; Translations; United States National Institutes of Health; Variant; Work; abstracting; amino acid metabolism; antimicrobial; arm; base; fitness; in vivo; inhibitor/antagonist; medical schools; mutant; pathogen; public health relevance; response; sensor; single molecule; single-molecule FRET; stem; tool

 DESCRIPTION (provided by applicant): Abstract Over the past decade, RNA-mediated regulation has emerged as a central theme in gene expression. A common control mechanism of Gram-positive bacteria is the riboswitch. In riboswitches, a sensor domain is mechanistically coupled to a regulatory domain composed of mutually exclusive terminator/anti-terminator RNA structure elements that either attenuate or increase translation/transcription. A particularly widespread type of riboswitch is the "T box", a tRNA-actuated switch whose default state is to attenuate transcription of essential genes encoding aminoacyl-tRNA synthetases, amino acid biosynthetic enzymes, and amino acid transport machinery. High ratios of charged-to-uncharged tRNA maintain the switch in the off state and suppress gene expression. The performance of the riboswitch is subject to modulation by multiple factors, including nucleotide sequence variations around conserved structure elements and tRNA base modification, and is crucial to the fitness of the cell. In this proposal, we will (1) identify the global conformationsand structural details of model tRNA-riboswitch complexes using SAXS and solution NMR methods; (2) develop a kinetic model of the T box riboswitch mechanism that includes variables such as tRNA modification and aminoacylation state using single molecule FRET techniques; and (3) determine in vivo the effects of tRNA modification and natural sequence variation on T box regulatory efficiency using direct RNA measurement assays. For the first time perhaps for any riboswitch, we will establish a mechanistic framework for riboswitch function that quantitatively connects local sequence-dependent contributions with global dynamics and T box efficiency. In addition, this project will broaden our knowledge of the contributions of tRNA nucleotide modification to gene regulation and cell fitness of Gram-positive bacteria. Finally, the T box riboswitch offers a unique platform on which to engineer genetic tools to interrogate the metabolic state of bacterial cells.

 描述（由适用提供）：在过去的十年中，RNA介导的调节已成为基因表达的中心主题。革兰氏阳性细菌的常见控制机制是核糖开关。在Riboswitch中，传感器结构域是机械耦合到由相互排他性终结器/抗终端RNA结构元件组成的调节域，该元素可以减弱或增加翻译/转录。一种特别宽的核糖开关类型是“ T盒”，“ T盒”是一种tRNA驱动的开关，其默认状态是减弱编码氨基酰基-TRNA合酶，氨基酸生物合成酶和氨基酸转运机械的基本基因的转录。高电荷与胸腔的tRNA比率保持在OFF状态下的开关并抑制基因表达。核糖开关的性能受到多个因素的调节，包括核苷酸序列序列变化，周围的结构元素和tRNA碱基修饰，对细胞的适应性至关重要。在此提案中，我们将使用SAXS和解决方案NMR方法确定模型trna-riboswitch复合物的全局会议和结构细节； （2）开发了T盒核糖开关机制的动力学模型，该模型包括使用单分子FRET技术等变量，例如tRNA修饰和氨基酰化状态； （3）使用直接RNA测量测定法确定体内tRNA修饰和自然序列变化对T框调节效率的影响。对于任何Riboswitch的首次可能性，我们将建立一个机械框架，以定量地将局部序列依赖性贡献与全局动力学和T盒效率连接起来。此外，该项目将扩大我们对tRNA核苷酸对基因调节和革兰氏阳性细菌细胞适应性的贡献的了解。最后，T盒Riboswitch提供了一个独特的平台，可以在该平台上设计遗传工具来询问细菌细胞的代谢状态。