Regulation and function of bacterial hibernating 100S ribosome

项目总结 核糖体冬眠是细菌和真核生物用来阻止翻译的一种保守机制。 并延长生物的寿命。最近对各种细菌物种的研究，包括致病菌 金黄色葡萄球菌，已经提供了令人信服的证据，证明了冬眠100s核糖体在 除了阻止翻译起始外，还保护核糖体池免受损害。我们发现金黄色葡萄球菌 缺乏冬眠促进因子(HPF)的核糖体被3‘-5’核酸外切酶迅速降解 R和其他迄今未知的核糖核酸酶。在我们未发表的工作中，我们分离出了一种额外的核糖核酸酶 挽救金黄色葡萄球菌核糖体丢失的突变体。令人惊讶的是，我们发现核糖体并不是唯一的 金黄色葡萄球菌HPF的靶标；相反，HPF可以与一种生物学活性未知的细胞质蛋白相互作用， 从而减少冬眠的100S核糖体的丰度。我们进一步证明了HPF是受限的 在快速生长期间的特定亚细胞定位，提供了一个罕见的可能的HPF分离的一瞥 主动翻译核糖体。在这项提案中，我们将采取高度多学科的方法 由结构生物学、组学、细菌遗传学、生物化学和高分辨率显微镜组成 实现以下目标：(1)确定HPF保护核糖体的分子机制 核糖核裂解。(2)确定以前未发现的HPF的体外作用。(三)确定 HPF的时空局部化如何避免翻译冲突。HPF和核糖核酸酶R在进化上 医院内革兰氏阳性和革兰氏阴性细菌的保守毒力因子，完成这些 AIMS将为抗击顽固性感染的创新对策提供重要的机械性见解 通过扰乱冬眠核糖体的生物发生和周转。

PROJECT SUMMARY Ribosome hibernation is a conserved mechanism used by both bacteria and eukaryotes to prevent translation and to extend organismal lifespan. Recent studies from various bacterial species, including pathogenic Staphylococcus aureus, have provided compelling evidence for a critical role of hibernating 100S ribosomes in protecting the ribosomal pool from damage, in addition to blocking translational initiation. We found that S. aureus ribosomes lacking hibernation-promoting factor (HPF) are rapidly degraded by the 3’-5’ exonuclease RNase R and other hitherto unknown ribonucleases. In our unpublished work, we isolated an additional ribonuclease mutant that rescues the loss of ribosomes in S. aureus. Surprisingly, we found that ribosomes are not the only target of S. aureus HPF; instead, HPF could interact with a cytoplasmic protein of unknown biological activity, thereby reducing the abundance of hibernating 100S ribosomes. We further demonstrated that HPF is restricted to a specific subcellular localization during rapid growth, providing a rare glimpse of possible HPF segregation from actively translating ribosomes. In this proposal, we will undertake a highly multidisciplinary approach consisting of structural biology, omics, bacterial genetics, biochemistry and high-resolution microscopy to achieve the following goals: (1) Determine the molecular mechanisms by which HPF protects ribosomes from ribonucleolytic cleavage. (2) Determine the previously undiscovered extraribosomal role of HPF. (3) Determine how the spatiotemporal localization of HPF avoids translation conflicts. HPF and RNase R are evolutionarily conserved virulence factors among nosocomial gram-positive and gram-negative bacteria, completion of these aims will provide significant mechanistic insight into innovative counterstrategies to combat recalcitrant infections by perturbing the biogenesis and turnover of hibernating ribosomes.