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拯救的功能 并针对这些途径开发新的抗生素。拟议项目的总体目标是 识别核糖体特异性所需的翻译机制组件之间的相互作用 拯救以及在什么条件下需要不同的核糖体拯救系统。这个问题的核心假设是 核糖体内部以及核糖体与其他翻译因子之间的特异性相互作用 是核糖体拯救所独特需要的，并且在环境条件下替代拯救系统是至关重要的。 导致RNA损伤的条件。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，这将使新抗生素的开发成为可能。中央 将通过追求以下具体目标来检验假设：1）确定所需的分子相互作用 对于反式翻译，2）确定ArfT如何与RF1或RF2结合拯救核糖体，以及3） 确定为什么需要替代核糖体拯救系统。已发表的工作和初步数据 确定了反式翻译的小分子抑制剂，并在该资助的第一个资助期内开展工作 确定了它们的分子靶点目标1将使用生化和突变分析来确定原因 这些靶点对于反式翻译和靶点如何被抑制剂结合破坏是重要的。我们将 我在目标2中使用了结构和生化实验来确定一种新的替代核糖体的机制 拯救途径，ArfT，可以募集RF1或RF2到不停止的核糖体。我们的初步数据显示 在新月柄杆菌中需要替代核糖体拯救因子ArfB的条件下，即使当 翻译是功能性的。我们将确定ArfB要求的分子基础，并确定 在其他细菌的类似挑战下，需要其他替代的核糖体拯救因子。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， 这里提出的工作是有意义的，因为它将描述核糖体拯救的生理要求 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 identify interactions among components of the translation machinery that are specifically required for ribosome rescue and under what conditions different ribosome rescue systems are required. The central hypothesis of this work is that specific interactions within the ribosome and between the ribosome and other translation factors are uniquely required for ribosome rescue and that alternative rescue systems are critical under environmental conditions that cause RNA damage. 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 trans-translation, 2) determine how ArfT rescues ribosomes in conjunction with either RF1 or RF2, and 3) determine why alternative ribosome rescue systems are required. Published work and preliminary data have identified small molecule inhibitors of trans-translation, and work in the first funded period of this grant identified their molecular targets. Biochemical and mutational analyses will be used in Aim 1 to determine why these targets are important for trans-translation and how the targets are disrupted by inhibitor binding. We will used structural and biochemical experiments in Aim 2 to determine the mechanism of a new alternative ribosome rescue pathway, ArfT, that can recruit either RF1 or RF2 to non-stop ribosomes. Our preliminary data identified conditions where the alternative ribosome rescue factor ArfB is required in Caulobacter crescentus, even when trans-translation is functional. We will determine the molecular basis for the ArfB requirement and determine of other alternative ribosome rescue factors are required under similar challenges in other bacteria. 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.