Rescue and repair of stalled ribosome damaged by ribosome-specific ribotoxins

被核糖体特异性核毒素损坏的停滞核糖体的拯救和修复

• 批准号: 10467347
• 负责人: Raven H Huang
• 金额: $ 31.84万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467347
• 关键词: Abbreviations; Antidotes; Archaea; Attenuated; Bacteria; Bacterial Proteins; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Assay; Biological Phenomena; Biological Process; Biology; C-terminal; Capnocytophaga; Cell Death; Cells; Chimeric Proteins; Complex; Conflict (Psychology); Coupled; Cryoelectron Microscopy; Data; Effectiveness; Engineering; Enterobacter cloacae; Escherichia coli; Eukaryota; Event; Genes; Homologous Gene; Human; In Vitro; Introns; Lead; Microbiology; Molecular; Operon; Organism; Pathway interactions; Prevention; Proteins; RNA; RNA Splicing; Reaction; Research; Ribosomal RNA; Ribosomes; Ricin; Site; Structure; System; Tail; Testing; Time; Toxic effect; Toxin; Transfer RNA; Translations; Virus; X-Ray Crystallography; biological systems; cell killing; colicin; experimental study; genome-wide; human disease; in vivo; insight; member; oral pathogen; pathogenic bacteria; release factor; repaired; small molecule inhibitor; structural biology; transcriptome sequencing; weapons

Project Summary/Abstract In biology, there are two fundamental conflicts: the conflicts between organisms and the conflicts between organisms and viruses. During the conflicts, a variety of offensive and defensive weaponry are employed by organisms and viruses. While the molecular mechanisms of the conflicts between organisms and viruses have been extensively studied, significantly less have been carried out with the conflicts between organisms. During the conflicts between organisms, protein toxins are frequently employed as the offensive weapons. Among them, ribotoxins constitutes one of the biggest groups. In fact, colicin E3 was the first ribotoxin to be characterized 50 years ago. It makes a single but precise cut of 16S rRNA in the decoding center of bacterial ribosome, resulting in stalled ribosome and eventual cell death. Over the last half of a century, it is unclear whether there exists a biological system that is able to reverse the ribosomal damage by colicin E3 to allow cell to survive. Employing approaches of bioinformatics, biochemistry, structural biology, and microbiology, we have uncovered a bacterial two-component system, RtcB and PrfH, as the antidote of colicin E3. Specifically, bacterial PrfH recognizes the damaged and stalled ribosome and performs ribosomal rescue. This is followed by RtcB repairing the damaged 30S ribosomal subunit. The sequential events described above are supported by abundant preliminary data from both our in vitro and in vivo studies. In this application, we plan to significantly expand our preliminary studies to systematically characterize the rescue and repair of bacterial ribosome with specific damage in the decoding center with the following three main aims: 1) We will provide insight into bacterial PrfH recognizing and rescuing the damaged and stalled 70S ribosome in vitro; 2) We will biochemically and structurally characterize bacterial RtcB in vitro, with the emphasis of PrfH-coupled RtcB repairing the damaged 30S ribosomal subunit; and 3) We will elucidate in vivo biological functions of RtcB-PrfH using an in vivo attenuated RNA damage system we have developed.

项目总结/摘要 在生物学中，有两种基本的冲突：生物体之间的冲突和 有机体和病毒之间的联系在冲突期间，各种进攻性和防御性武器 被生物体和病毒利用。虽然生物体之间冲突的分子机制 和病毒已经被广泛研究，但在冲突中进行的研究却少得多。 在生物体之间。在生物体之间的冲突中，蛋白质毒素经常被用作 攻击性武器其中，核毒素是最大的一类。事实上，大肠杆菌素E3 50年前发现的第一种核糖毒素它对16 S rRNA进行单一但精确的切割， 细菌核糖体的解码中心，导致停滞的核糖体和最终的细胞死亡。在过去 半个世纪以来，人们还不清楚是否存在一种生物系统，能够逆转核糖体 大肠杆菌素E3的破坏，使细胞存活。运用生物信息学、生物化学、 结构生物学和微生物学，我们已经发现了一个细菌双组分系统，RtcB和PrfH， 大肠杆菌素E3的解毒剂具体来说，细菌PrfH识别受损和停滞的核糖体， 进行核糖体拯救。随后是RtcB修复受损的30 S核糖体亚基。的 上述连续事件得到了来自我们的体外和体内的大量初步数据的支持。 体内研究。在这项申请中，我们计划大大扩展我们的初步研究， 表征在解码中心具有特异性损伤的细菌核糖体的拯救和修复， 以下三个主要目标：1）我们将提供深入了解细菌PrfH识别和拯救 2）我们将在体外对70 S核糖体进行生物化学和结构表征， 体外RtcB，重点是PrfH偶联的RtcB修复受损的30 S核糖体亚基;和3） 我们将使用体内减弱的RNA损伤系统阐明RtcB-PrfH的体内生物学功能 我们已经开发了。