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

项目摘要/摘要 在生物学中，有两个根本的冲突：有机体之间的冲突和生物之间的冲突 在有机体和病毒之间。在冲突期间，各种攻防武器装备 被有机体和病毒利用的。而生物体之间冲突的分子机制 人们对病毒进行了广泛的研究，但在冲突中进行的研究明显较少 在有机体之间。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，核毒素构成了最大的群体之一。事实上，粘菌素E3是 这是50年前发现的第一种核毒素。它对16S rRNA进行了一次但精确的切割 细菌核糖体的解码中心，导致核糖体停滞并最终导致细胞死亡。在过去的几年里 半个世纪以来，尚不清楚是否存在一种能够逆转核糖体的生物系统 粘菌素E3的破坏，使细胞得以存活。利用生物信息学、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， 作为粘菌素E3的解毒剂。具体来说，细菌PrfH识别受损和停滞的核糖体，并 执行核糖体抢救。其次是RtcB修复受损的30S核糖体亚基。这个 上述顺序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 活体研究。在这项申请中，我们计划显著扩展我们的初步研究，以系统地 在解码中心表征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)我们将深入了解细菌性PrfH识别和拯救 70s核糖体的体外损伤和停滞；2)我们将对细菌进行生化和结构表征 RtcB在体外，重点是PrfH偶联的RtcB修复受损的30S核糖体亚基； 我们将使用体内减弱的RNA损伤系统来阐明RtcB-PrfH的体内生物学功能 我们已经开发了。

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.