Regulation and function of bacterial hibernating 100S ribosome

项目摘要 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 并延长生物体的寿命。最近对各种细菌物种的研究，包括致病性 金黄色葡萄球菌，提供了令人信服的证据，证明冬眠的100 S核糖体在 除了阻断翻译起始外，还保护核糖体库免受损伤。我们发现S.金黄色 缺乏冬眠促进因子（HPF）的核糖体被3 '-5'核酸外切酶RNase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，我们分离出一种额外的核糖核酸酶， 突变体，挽救了S中核糖体的损失。金黄色。令人惊讶的是，我们发现核糖体不是唯一的 S的目标。金黄色葡萄球菌HPF;相反，HPF可以与未知生物活性的细胞质蛋白相互作用， 从而减少冬眠的100 S核糖体的丰度。我们进一步证明了HPF是受限制的， 在快速生长过程中的一个特定的亚细胞定位，提供了一个罕见的可能HPF分离一瞥 核糖体的主动翻译。在这一建议中，我们将采取高度多学科的方法， 包括结构生物学、组学、细菌遗传学、生物化学和高分辨率显微镜， 实现以下目标：（1）确定HPF保护核糖体免受 核糖核裂解(2)确定以前未发现的HPF的核糖体外作用。(3)确定 HPF的时空定位如何避免翻译冲突。HPF和RNase R在进化上 保守的毒力因子之间的医院革兰氏阳性和革兰氏阴性细菌，完成这些 aims将提供重要的机制洞察到创新的对策，以打击寄生虫感染 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 In our unpublished work, we isolated an additional ribonuclease mutant that rescues the loss of ribosomes in S. aureus.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.