Role of nucleoside modifications in tRNA surveillance in prokaryotes

核苷修饰在原核生物 tRNA 监测中的作用

• 批准号: 1412379
• 负责人: Valerie de Crecy-Lagard
• 金额: $ 69万
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1412379&HistoricalAwards=false
• 关键词: Role; nucleoside; modifications; tRNA; surveillance

All life requires the active participation of a class of RNA molecules known as transfer RNA (tRNA) and many biological processes act to ensure the proper functioning of this tRNA pool by removing damaged or defective tRNA molecules before they can cause harm to the cell. This project is focused on discovering new pathways for protecting bacterial cells from damaged tRNA, as might occur occasionally due to mistakes made by the enzymes that are responsible for producing tRNA, or due to growth in stressful conditions. Although some of these pathways have been identified in eukaryotes, the fate of these damaged tRNAs is not completely understood in Bacteria. In addition, the research will determine the function of potentially novel tRNA metabolism enzymes that were identified by integrative data mining and appear likely to play important but unknown roles in tRNA metabolism. The project will train one graduate student and two post-doctoral fellows to use cutting-edge multidisciplinary approaches to solve fundamental biological problems, and will develop three classroom-ready online modules using biological data-mining tools for high school teachers. The goal of this research is to discover novel regulatory processes involving tRNA turnover in prokaryotes and to understand the role of tRNA modifications in these processes. This interdisciplinary project integrates computational analyses with innovative technologies that encompass bacterial genetics, RNA biochemistry, bioanalytical chemistry and genomics and the use of novel systems-level analysis tools to survey the modification and cleavage profiles of all tRNAs in the cell. The research will analyze the role of modifications as determinants for tRNA integrity in Bacteria by conducting screens for synthetic growth phenotypes, combining deletions in tRNA modification genes under different stress conditions and measuring tRNA levels, modification status and fragment population in strains lacking specific modifications. In addition, preliminary comparative genomic analysis has linked bacterial enzymes related to tRNA metabolism, but whose precise biological function is unknown, to both tRNA modification and stress resistance, suggesting they participate in novel mechanisms involving tRNA degradation and/or repair. This hypothesis will be tested by analyzing tRNA pro-files in E. coli derivatives lacking the corresponding genes and by identifying specific RNA substrates for these orphan RNA metabolism enzymes.

所有生命都需要一类被称为转移RNA(TRNA)的RNA分子的积极参与，许多生物过程通过在tRNA分子对细胞造成伤害之前将其移除，以确保tRNA池的正常功能。该项目的重点是发现保护细菌细胞免受tRNA损伤的新途径，这种损伤可能偶尔发生，因为负责生产tRNA的酶出错，或者由于在应激条件下的生长。虽然这些途径中的一些已经在真核生物中被发现，但这些受损的tRNAs在细菌中的命运并不完全清楚。此外，这项研究将确定潜在的新型tRNA代谢酶的功能，这些酶是通过综合数据挖掘确定的，似乎可能在tRNA代谢中发挥重要但未知的作用。该项目将培训一名研究生和两名博士后研究员使用尖端的多学科方法解决基本的生物学问题，并将为高中教师开发三个使用生物学数据挖掘工具的课堂上随时可用的在线模块。本研究的目的是发现原核生物中涉及tRNA周转的新的调控过程，并了解tRNA修饰在这些过程中的作用。这一跨学科项目将计算分析与创新技术相结合，包括细菌遗传学、RNA生物化学、生物分析化学和基因组学，并使用新的系统级分析工具来调查细胞中所有tRNA的修饰和切割情况。这项研究将分析修饰作为细菌tRNA完整性决定因素的作用，方法是对合成生长表型进行筛选，结合不同胁迫条件下tRNA修饰基因的缺失，并测量缺乏特定修饰的菌株中的tRNA水平、修饰状态和片段数量。此外，初步的比较基因组分析表明，细菌酶与tRNA代谢相关，但其确切的生物学功能尚不清楚，与tRNA修饰和抗逆性有关，表明它们参与了涉及tRNA降解和/或修复的新机制。这一假设将通过分析缺乏相应基因的大肠杆菌衍生物中的tRNA图谱，以及通过识别这些孤儿RNA代谢酶的特定RNA底物来验证。