Physiology of ribosome rescue in bacteria

总结 核糖体拯救途径在整个细菌中是保守的，但是这些途径对于 生理学不被理解。这个项目的长期目标是了解核糖体的功能 拯救途径，并针对这些途径开发新的抗生素。拟议项目的总体目标 是使用核糖体拯救抑制剂作为探针，以了解核糖体拯救在原子，分子， 细胞水平。这项工作的中心假设是，核糖体拯救是普遍需要的， 维持细菌中的蛋白质合成能力，并且拯救的抑制剂靶向 翻译机器。进行拟议研究的理由是，它将确定为什么 核糖体拯救在细菌中是保守的，并且将使得能够开发新的抗生素。中央 我们将通过以下具体目标来检验这一假设：1）确定分子间的相互作用 2）确定为什么核糖体拯救对于细菌生理学是重要的，以及3） 发现核糖体拯救的新机制。已发表的工作和初步数据已经确定了四个 抑制核糖体拯救的化学相关小分子家族。这些化合物将用于 目的1 a）鉴定负责抑制核糖体拯救的分子靶标和结合位点，和B） 确定与靶分子的相互作用如何阻止核糖体拯救而不是翻译。我们将使用 小分子抑制剂作为化学生物学工具，以检查核糖体抑制的三种结果 拯救：核糖体的状态，这是核糖体拯救的直接目标;和蛋白质组学和 当核糖体拯救成为生长限制时，转录组学反应。Aim的工作假设 3是在所有细菌中至少需要一个核糖体拯救系统。这一假设将通过使用 Tn-seq鉴定枯草芽孢杆菌和土拉弗朗西斯菌中的替代核糖体拯救因子， 没有tmRNA的物种不包含已知的备份系统之一。使用 用于化学生物学实验以探测核糖体拯救的小分子抑制剂是高度创新的， 本文提出的工作是有意义的，因为它将阐明核糖体的生理需要。 拯救细菌中的途径，并确定如何抑制这些途径。

SUMMARY Ribosome rescue pathways are conserved throughout bacteria, but the reason these pathways are important for physiology is not understood. The long-term goal of this project is to understand the function of ribosome rescue pathways and to target these pathways for new antibiotics. The overall objective of the proposed project is to use ribosome rescue inhibitors as probes to understand ribosome rescue at the atomic, molecular, and cellular levels. The central hypothesis of this work is that ribosome rescue is universally required for maintaining protein synthesis capacity in bacteria and that inhibitors of rescue target conserved components of the translation machinery. The rationale for pursuing the proposed research is that it will determine why ribosome rescue is conserved in bacteria and will enable development of new antibiotics. The central hypothesis will be tested by pursuing the following specific aims: 1) identify the molecular interactions required for ribosome rescue, 2) determine why ribosome rescue is important for bacterial physiology and 3) discover new mechanisms for ribosome rescue. Published work and preliminary data have identified four chemically related families of small molecules that inhibit ribosome rescue. These compounds will be used in Aim 1 to a) identify the molecular targets and binding sites responsible for inhibiting ribosome rescue and b) determine how interactions with target molecules block ribosome rescue but not translation. We will use the small molecule inhibitors as chemical biology tools to examine three outcomes from inhibition of ribosome rescue: the status of ribosomes, which are the direct target of ribosome rescue; and the proteomic and transcriptomic responses when ribosome rescue becomes limiting for growth. The working hypothesis for Aim 3 is that at least one ribosome rescue system is required in all bacteria. This hypothesis will be tested by using Tn-seq to identify alternative ribosome rescue factors in Bacillus subtilis and Francisella tularensis, the only species shown to survive without tmRNA that do not contain one of the known backup systems. The use of small molecule inhibitors for chemical biology experiments to probe ribosome rescue is highly innovative, and the work proposed here is significant because it will delineate the physiological requirement for ribosome rescue pathways in bacteria and identify how these pathways can be inhibited.