A bioanalytical platform for interrogating the systems biology of tRNA modifications: Application to defining translational control mechanisms in bacterial stress responses

用于探究 tRNA 修饰的系统生物学的生物分析平台：应用于定义细菌应激反应中的翻译控制机制

• 批准号: 1308839
• 负责人: Peter Dedon
• 金额: $ 45万
• 依托单位: Massachusetts Institute of Technology
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1308839&HistoricalAwards=false
• 关键词: bioanalytical; platform; interrogating; systems; biology

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Professor Peter Dedon from the Massachusetts Institute of Technology to characterize a newly discovered, translation-based mechanism of bacterial stress response, in which stress causes reprogramming of dozens of tRNA modifications that regulate the translation of codon-biased transcripts to achieve selective expression of critical proteins. The studies will be initiated by comprehensively characterizing modified ribonucleosides in tRNA in Pseudomonas aeruginosa (PA) using a mass spectrometry-based platform developed in these labs. Following exposure of PA to hydrogen peroxide, changes in the spectrum of modified ribonucleosides will be quantified and modifications found to increase significantly under stress will be localized within specific tRNA molecules. Stress-altered wobble modifications will then be used to predict anticodon-codon interactions involved in selective translation of specific mRNAs for response and adaptation proteins, with proteomic validation of stress-induced changes in protein levels. Finally, a transposon mutant library will be used to define the biosynthetic pathways for stress-altered modifications by screening tRNA modification spectral changes in PA mutants selected by homology to other prokaryotes. The proposed studies will provide new insights into the translational mechanisms governing bacterial responses to environmental conditions, and the detailed molecular mechanisms that link translation to cell phenotype. The goal of these studies is to see if bacteria use a novel mechanism of cell survival and adaptation recently discovered in yeast and human cells. Using technology for quantifying the dozens of modified ribonucleotide building blocks of transfer RNA (tRNA) - the molecules controlling protein synthesis in all living organisms - it was discovered that stressed cells "reprogram" their tRNA modifications in a way that leads to selective production of proteins that are required to survive to the stress. The generality of this model will now be tested in the bacterium, Pseudomonas aeruginosa, to begin to understand how translational control mechanisms affect the ability of this important pathogenic microbe to survive and adapt during infection. The methods developed here will provide microbiologists with tools to systematically study RNA modifications in any type of bacterium, in principle, with potential broad impact on the basic science of cell and molecular biology. The studies will also provide students and scientists with specialized training in analytical technology and in a new fundamental mechanism of cell biology, while an outreach program will engage high school biology students with hands-on activities in cell culture, RNA isolation and mass spectrometry.

有了这个奖项，化学系的生命过程计划的化学正在资助来自马萨诸塞州理工学院的Peter Dedon教授，以表征一种新发现的，基于防御的细菌应激反应机制，其中应激导致数十种tRNA修饰的重新编程，这些修饰调节密码子偏向的转录本的翻译，以实现关键蛋白质的选择性表达。这些研究将通过使用这些实验室开发的基于质谱的平台全面表征铜绿假单胞菌（PA）中tRNA中的修饰核糖核苷来启动。在PA暴露于过氧化氢后，修饰的核糖核苷谱的变化将被量化，并且发现在应力下显著增加的修饰将定位于特定的tRNA分子内。压力改变的摆动修饰，然后将用于预测反密码子-密码子的相互作用参与选择性翻译的特定mRNA的反应和适应蛋白，与蛋白质组学验证的压力诱导的蛋白质水平的变化。最后，转座子突变体库将被用来定义的生物合成途径的压力改变的修改，通过筛选tRNA修饰光谱变化的PA突变体的同源性选择其他原核生物。拟议的研究将为细菌对环境条件的反应的翻译机制以及将翻译与细胞表型联系起来的详细分子机制提供新的见解。 这些研究的目的是观察细菌是否使用最近在酵母和人类细胞中发现的细胞存活和适应的新机制。使用定量技术来量化转移RNA（tRNA）的数十种修饰的核糖核苷酸构建块-控制所有生物体中蛋白质合成的分子-发现应激细胞以一种导致选择性生产在应激中生存所需的蛋白质的方式“重新编程”其tRNA修饰。现在将在细菌铜绿假单胞菌中测试该模型的通用性，以开始了解翻译控制机制如何影响这种重要病原微生物在感染期间存活和适应的能力。这里开发的方法将为微生物学家提供系统研究任何类型细菌中RNA修饰的工具，原则上，对细胞和分子生物学的基础科学具有潜在的广泛影响。这些研究还将为学生和科学家提供分析技术和细胞生物学新基本机制的专业培训，而一个外展计划将使高中生物学学生参与细胞培养，RNA分离和质谱分析的实践活动。