Resolving Structure and Mechanism of tRNA-actuated riboswitches

解析 tRNA 驱动核糖开关的结构和机制

基本信息

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
项目类别：
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

项目摘要

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介导的调控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，在核糖开关中，传感器结构域机械地偶联。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，可减弱或增强翻译/转录核糖开关的一种特别广泛的类型是“T 盒”，一种 tRNA 驱动的。开关的默认状态是减弱编码氨酰基 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，高比例的带电 tRNA 与不带电 tRNA 维持开关处于关闭状态并抑制基因表达。核糖开关的功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，这对于细胞的适应性至关重要。 (1) 使用 SAXS 和溶液 NMR 方法识别模型 tRNA-核糖开关复合物的整体构象和结构细节；(2) 使用单分子 FRET 技术开发 T 盒核糖开关机制的动力学模型，其中包括 tRNA 修饰和氨酰化状态等变量。 (3) 使用直接 RNA 测量测定法确定体内 tRNA 修饰和自然序列变异对 T 盒调节效率的影响，这对于任何核糖开关来说可能是第一次。此外，该项目还将拓宽我们对 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。，T 盒核糖开关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。