Physiology of ribosome rescue in bacteria

细菌核糖体拯救的生理学

基本信息

批准号：
9382125
负责人：
KENNETH C KEILER
金额：
$ 42.72万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2021-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9382125
关键词：
Antibiotics Bacillus subtilis Bacteria Bacterial Genome Bacterial Physiology Binding Binding Sites Biochemical Biological Biological Assay Biology Cells Chemicals Data Databases Development Drug Targeting Family Francisella tularensis Genes Genetic Goals Growth Health Human Hydrolysis Knowledge Messenger RNA Molecular Molecular Target Multi-Drug Resistance Outcome Pathogenicity Pathway interactions Pharmacology Physiological Physiology Process Property Protein Biosynthesis Proteomics Publishing Research Ribosomal Interaction Ribosomes Role SmpB protein Solid System Terminator Codon Testing Translating Translations Virulence Work bacterial resistance base drug development experimental study improved inhibitor/antagonist innovation insight mRNA Transcript Degradation pathogen peptidyl-tRNA polypeptide response ribosome profiling small molecule small molecule inhibitor tmRNA tool transcriptomics

项目摘要

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.

概括核糖体救援途径在整个细菌中都保留了，但是这些途径对于生理学尚不理解。该项目的长期目标是了解核糖体的功能营救途径并针对这些新抗生素的途径。拟议项目的总体目标是使用核糖体救援抑制剂作为探针，以了解原子，分子和细胞水平。这项工作的中心假设是核糖体救援是普遍需要的维持细菌中的蛋白质合成能力和救援靶标的抑制剂的保守成分翻译机械。追求拟议的研究的理由是，它将决定为什么核糖体救援是在细菌中保守的，将能够开发新的抗生素。中央假设将通过追求以下特定目的来检验：1）确定分子相互作用核糖体营救所需，2）确定为什么核糖体救援对细菌生理很重要，而3）发现核糖体救援的新机制。已发布的工作和初步数据已经确定了四个抑制核糖体营救的小分子的化学相关家族。这些化合物将用于目标1 a）识别负责抑制核糖体救援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但不能翻译。我们将使用小分子抑制剂作为化学生物学工具，可以检查核糖体的抑制作用三个结果救援：核糖体的状态，这是核糖体营救的直接靶标；以及蛋白质组学和当核糖体救援成为生长的限制时，转录组反应。目标的工作假设 3是所有细菌中至少需要一个核糖体救援系统。该假设将通过使用 TN-Seq确定枯草芽孢杆菌和弗朗西斯菌的替代性核糖体救援因子，唯一没有TMRNA生存的物种，这些物种不包含一个已知的备份系统之一。使用用于化学生物学实验的小分子抑制剂以探测核糖体救援具有很高的创新性，并且这里提出的工作很重要，因为它将描述核糖体的生理要求营救细菌中的途径，并确定如何抑制这些途径。