Elucidating the role of peptidyl-tRNA hydrolase activities in translation regulation

蛋白质的合成是维持所有生物体生命的基本活动。活细胞内的蛋白质合成过程通常是高效而顺利的。然而，偶尔，尤其是在压力条件下，蛋白质合成机制遇到问题和停滞。幸运的是，细胞配备了质量控制系统，致力于克服蛋白质合成中的问题。质量控制体系的多个组成部分和作用机制还远远没有被充分描述和理解。在本项目中，将研究细菌中一个以前未知的关键蛋白质合成质量控制系统的细胞因素和分子机制。由此产生的新知识将为对抗细菌感染的新策略提供信息。这些研究活动将被纳入培养高中、大学、研究生和博士后的教育计划中。研究结果将通过在地方、国家和国际会议上的报告以及在同行评议期刊上的出版物向科学界通报。人们一直认为肽基tRNA水解酶（Pth）对细菌至关重要，因为它能够从肽-tRNA复合物中回收转运RNA （tRNA）分子，这些分子在翻译失败的循环中从核糖体中分离出来。最近，人们发现Pth能够切割核糖体上的肽基trna，这些核糖体已经停止制造具有问题序列的蛋白质。这一新发现的Pth参与翻译质量控制揭示了这种酶的重要性的原因尚不完全清楚。在这个项目中，核糖体分析（Ribo-Seq），一种揭示核糖体全基因组位置的方法，将有助于确定两种模式细菌（大肠杆菌和枯草芽孢杆菌）中的基因位点，其中Pth作为核糖体努力翻译特定序列的拯救因子。此外，生物化学和结构方法将用于揭示pth驱动核糖体救援的机制原理。该项目将促进我们对蛋白质合成质量控制策略的理解。该项目的研究结果可能指导未来的研究，以设计新型抗生素和治疗与蛋白质合成缺陷相关的疾病。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Synthesis of proteins is a fundamental activity that sustains the life of all organisms. The process of synthesizing proteins inside living cells normally occurs efficiently and smoothly. However, occasionally, and even more so under stress conditions, the protein synthesis machinery encounters problems and stalls. Fortunately, cells are equipped with quality control systems dedicated to overcoming problems in protein synthesis. The multiple components and mechanisms of action of the quality control systems are far from being fully characterized and understood. In this project, the cellular factors and molecular mechanism of a previously unknown critical protein synthesis quality control system in bacteria will be studied. The resulting new knowledge will inform new strategies for combating bacterial infections. The research activities will be incorporated in educational programs to train students from high school, college, and graduate programs, as well as postdoctoral researchers. The research findings will be communicated to the scientific community through presentations at local, national, and international meetings and by publications in peer-reviewed journals. It has been assumed that the enzyme peptidyl-tRNA hydrolase (Pth) is essential for bacteria because of its ability to recycle transfer RNA (tRNA) molecules from peptide-tRNA complexes that dissociate from the ribosome during abortive cycles of translation. Recently, it has come to light that Pth is capable of cleaving peptidyl-tRNAs on ribosomes that have stopped making proteins with problematic sequences. This newly discovered involvement of Pth in translation quality control reveals that the reasons for the essentiality of this enzyme are yet to be fully understood. In this project, ribosome profiling (Ribo-Seq), an approach that reveals the positions of ribosomes genome-wide, will help identify the gene sites in two model bacteria (Escherichia coli and Bacillus subtilis) where Pth acts as a rescue factor for ribosomes struggling to translate specific sequences. In addition, biochemical and structural approaches will be used to reveal the mechanistic principles underlying Pth-driven ribosome rescue. The project will advance our understanding of strategies for protein synthesis quality control. The findings of the project may guide future research to design novel antibiotics and treatments for diseases associated with defective protein synthesis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.