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Physiology of ribosome rescue in bacteria

细菌核糖体拯救的生理学

基本信息

批准号：
9535410
负责人：
KENNETH C KEILER
金额：
$ 39.86万
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/9535410
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.

摘要核糖体拯救途径在细菌中是保守的，但这些途径对生理学还不为人所知。这个项目的长期目标是了解核糖体的功能。拯救这些途径，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物作为探针，在原子、分子和细胞水平。这项工作的中心假设是，核糖体拯救是普遍需要的在细菌中维持蛋白质合成能力和救援靶标的抑制剂保守的成分翻译机器。继续这项拟议研究的理由是它将确定为什么核糖体挽救在细菌中是保守的，并将使新抗生素的开发成为可能。中环假设将通过追求以下具体目标来检验：1)确定分子相互作用核糖体挽救所需，2)确定为什么核糖体挽救对细菌生理学很重要，3) 发现核糖体拯救的新机制。已发表的工作和初步数据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标1)确定抑制核糖体拯救的分子靶点和结合部位；b) 确定与目标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检测核糖体抑制的三种结果抢救：作为核糖体抢救的直接靶标的核糖体的状况；以及蛋白质组和当核糖体挽救对生长产生限制时的转录反应。AIM的工作假说 3是所有细菌都需要至少一个核糖体拯救系统。这一假设将通过使用 TN-SEQ在唯一的枯草芽孢杆菌和图拉氏弗朗西斯杆菌中寻找替代核糖体拯救因子显示了在没有tmRNA的情况下存活的物种，这些物种不包含已知的备份系统之一。对.的使用小分子抑制剂用于化学生物实验探测核糖体拯救具有很高的创新性，并且这里提出的工作具有重要意义，因为它将描绘核糖体的生理需求。拯救细菌中的通路，并确定如何抑制这些通路。