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

获得这一奖项的是，化学部生命过程化学计划资助麻省理工学院的Peter Dedon教授表征一种新发现的基于翻译的细菌应激反应机制，在这种机制中，应激导致数十种tRNA修饰重新编程，这些修饰调节偏向密码子的转录本的翻译，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学的平台来全面表征铜绿假单胞菌(PA)tRNA中的修饰核糖核苷来启动。在PA暴露于过氧化氢后，修饰核糖核苷的光谱变化将被量化，在应激条件下显著增加的修饰将定位于特定的tRNA分子。然后，应激改变的摆动修饰将被用于预测反密码子-密码子相互作用，涉及选择性地翻译特定的mRNAs以响应和适应蛋白质，并通过蛋白质组学验证应激诱导的蛋白质水平的变化。最后，转座子突变体文库将用于通过筛选与其他原核生物同源选择的PA突变体的tRNA修饰光谱变化来确定胁迫改变修饰的生物合成途径。拟议的研究将为控制细菌对环境条件的反应的翻译机制提供新的见解，以及将翻译与细胞表型联系起来的详细分子机制。这些研究的目标是看看细菌是否使用了最近在酵母和人类细胞中发现的一种新的细胞生存和适应机制。利用量化数十个修饰的转移RNA(TRNA)核糖核酸基(TRNA)的技术-所有生物中控制蛋白质合成的分子-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对其tRNA修饰进行重新编程。现在将在细菌铜绿假单胞菌中测试这一模型的普遍性，以开始了解翻译控制机制如何影响这种重要的病原微生物在感染期间的生存和适应能力。这里开发的方法将为微生物学家提供工具，从原则上系统地研究任何类型细菌的RNA修饰，可能对细胞和分子生物学的基础科学产生广泛影响。这些研究还将为学生和科学家提供分析技术和新的细胞生物学基本机制方面的专门培训，而一个外展项目将让高中生物学学生参与细胞培养、RNA分离和质谱学方面的实践活动。

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.