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（由申请人提供）：摘要在过去的十年中，RNA介导的调节已经成为基因表达的中心主题。革兰氏阳性菌的常见控制机制是核糖开关。在核糖开关中，传感器结构域机械地偶联到由相互排斥的终止子/抗终止子RNA结构元件组成的调节结构域，所述终止子/抗终止子RNA结构元件减弱或增加翻译/转录。一种特别广泛的核糖开关类型是“T盒”，一种tRNA激活的开关，其默认状态是减弱编码氨酰-tRNA合成酶、氨基酸生物合成酶和氨基酸转运机制的必需基因的转录。高比率的带电荷与不带电荷的tRNA使开关保持在关闭状态并抑制基因表达。核糖开关的性能受到多种因素的调节，包括保守结构元件周围的核苷酸序列变异和tRNA碱基修饰，并且对细胞的适应性至关重要。本论文的主要工作是：（1）用小角X射线散射和溶液核磁共振方法研究tRNA-核糖开关复合物的整体构象和结构细节;（2）用单分子FRET技术建立一个包含tRNA修饰和氨酰化状态等变量的T盒核糖开关机制的动力学模型;和（3）使用直接RNA测量分析在体内确定tRNA修饰和天然序列变异对T盒调节效率的影响。对于第一次，也许是任何核糖开关，我们将建立一个核糖开关功能的机制框架，定量连接本地序列依赖的贡献与全球动态和T盒效率。此外，该项目将拓宽我们对tRNA核苷酸修饰对革兰氏阳性菌基因调控和细胞适应性的贡献的认识。最后，T盒核糖开关提供了一个独特的平台，在这个平台上设计遗传工具来询问细菌细胞的代谢状态。