CAREER: Structure-function studies of bacterial toxin-ribosome complexes

原核生物已经进化出有效和高效的方法，通过全球调控反应来快速响应和生存不同的环境压力。这是通过调整细胞内代谢过程的速度来实现的，这些过程停止了必要的生长过程，如DNA复制、RNA转录和蛋白质合成。毒素-抗毒素(TA)模块在这种情况下促进细胞存活。此外，TA系统还与生物膜的形成、细菌在抗生素治疗过程中的持久性以及细菌的发病机制有关。这些不同的功能强调了确定如何识别细胞靶点以促进细胞生存的重要性。在营养紧张时期，细菌转向TA系统来微调基本的细胞过程。与炭疽、霍乱或白喉等经典细菌毒素截然不同的是，TA系统的毒素成分通常存在于与特定阻断其活性的抗毒素结合的细胞中。在应激条件下，抗毒素被降解，毒素被释放以发挥其作用，最常见的是核糖核酸酶对信使RNA的活性。杜纳姆实验室的研究集中在一种新的毒素蛋白上，这种蛋白只在与翻译核糖体结合时才能特异性地降解mRNA。这项研究需要使用集成的结构生物学和生化方法来回答RNA-蛋白质识别和催化的关键问题，并提供对毒素介导的mRNA降解的更深层次的了解。广泛影响：这项研究的更广泛影响包括学生参与这个项目的各个方面，从学习纯化核糖体，过度表达和纯化重组蛋白，RNA的体外转录和体外翻译试验。此外，学生将在结构生物学的前沿获得经验，学习解决大分子复合体所需的技术。PI将通过埃默里暑期本科生项目和S女子学院资助两名暑期本科生，以鼓励他们追求科学事业。最后，高中生和老师将在埃默里-佐治亚理工学院的暑期联合项目中获得实践经验，该项目旨在研究极端细菌核糖体。

ABSTRACTProkaryotes have evolved effective and efficient ways to rapidly respond to and survive diverse environmental stresses through global regulatory responses. This is achieved by adjusting rates of intracellular metabolic processes that halt essential growth processes such as DNA replication, RNA transcription and protein synthesis. Toxin-antitoxin (TA) modules facilitate cell survival during such times. In addition, TA systems have also been implicated in biofilm formation, bacterial persistence during antibiotic treatment and bacterial pathogenesis. These diverse functions underpin the importance of determining how recognition of cellular targets is achieved to facilitate cell survival. During times of nutritional stress, bacteria turn to TA systems to fine tune basic cellular processes. In distinct contrast to classic bacterial toxins such as anthrax, cholera or diptheria, the toxin component of a TA system is normally present in cells bound to an antitoxin that specifically blocks its activity. Under stress conditions, the antitoxin is degraded and the toxin released to exert its effect, most commonly ribonuclease activity against messenger RNA. The research in the Dunham laboratory focuses on a novel toxin protein that codon-specifically degrades mRNA, but only when bound to a translating ribosome. This research entails the use of an integrated structural biology and biochemical approach to answer key questions of RNA-protein recognition and catalysis and to provide a deeper understanding of the toxin mediated mRNA degradation.Broader Impacts: The Broader Impacts of this research include the involvement of students in all aspects of this project from learning to purify ribosomes, overexpression and purification of recombinant proteins, in vitro transcription of RNA and in vitro translation assays. In addition, students will gain experience at the forefront of structural biology, learning about techniques that are required to solve large macromolecular complexes. The PI will sponsor two summer undergraduate students through the Emory summer undergraduate program and from an all women?s college to encourage the pursuit of a scientific career. Lastly high school students and teachers will get hands-on experience in a joint Emory-Georgia Tech summer program designed to study extremophile ribosomes.