The role of HrpA in ribosome-associated quality control in E. coli

项目摘要/摘要 拉长的核糖体经常遇到导致核糖体停滞的障碍。停滞的核糖体 然后被救援因子靶向，以循环核糖体亚基，并靶向有缺陷的mrna和新生 抗腐败肽。这一过程对细菌的细胞适合性至关重要：它的丢失通常会导致 致病性、存活性或抗药性。在大肠杆菌中，核糖体停滞会导致碰撞，从而招募 SmrB内切酶切割相互碰撞的核糖体之间的mRNA。然后，这些核糖体被靶向 解救因子tmRNA.几条证据表明，大肠杆菌使用第二种途径来分解抗生素- 抑制核糖体转变为亚单位；然而，核糖体分裂因子尚未确定。DExH-Box RNA解旋酶HrpA降低了大肠杆菌对核糖体靶向抗生素的敏感性，这表明该蛋白具有 核糖体相关功能在抗生素耐药性中的作用。事实上，HrpA已被证明可以拯救停滞不前的核糖体 在报告mRNA和解决全球抗生素诱导的核糖体碰撞，表明它在 在翻译应激过程中清除堆积的核糖体。拟议的实验将探索HrpA是否 一种新的核糖体拯救因子，可以裂解停滞的核糖体。因为核糖体碰撞对核糖体至关重要 在酵母、哺乳动物细胞和大肠杆菌中的救援，Aim 1将确定核糖体碰撞是否也招募HrpA。 野生型和ΔhrpA细胞中基于报告的蛋白质读数将被用于测试hrpA是否拯救 核糖体与信使核糖核酸发生碰撞。为了确定HrpA是否优先与体内碰撞的核糖体相关联， 蔗糖密度梯度将用于确定HrpA是否与核糖体亚基、单一核糖体、 或者是用抑制核糖体的抗生素治疗的细胞裂解物中的多聚体发生碰撞。最后， 将使用核糖体图谱来探索核糖体位置上的HrpA和转录组范围内的核糖体碰撞 在抗生素处理的野生型和ΔhrpA细胞中。同一DExH-box家族中的两个酿酒酵母RNA解旋酶 因为HrpA参与核糖体分裂；因此，Aim 2将确定HrpA类似的分裂是否停滞 大肠杆菌中的核糖体。因为Hsp15蛋白优先与50S核糖体亚基结合 过早分裂核糖体，HrpA裂解活性将通过检测Hsp15在体内的沉积来检测 抗生素处理的野生型和ΔhrpA细胞中的蔗糖密度梯度。最后，将测试HrpA的能力 使用体外生化试验将停滞的核糖体分裂成亚基，其中分析了纯化的成分 蔗糖密度梯度。该方法将用于测定ATPase活性和精确底物 HrpA的识别。HrpA的特性将为了解大肠杆菌如何缓解肺炎的影响提供有价值的见解 核糖体靶向抗生素，使其成为未来抗菌化合物的潜在靶点 人类疾病的治疗。

Project Summary/Abstract Elongating ribosomes frequently encounter obstacles that cause ribosomes to stall. Stalled ribosomes are then targeted by rescue factors to recycle the ribosomal subunits and target the faulty mRNA and nascent peptide for decay. This process is critical for cellular fitness in bacteria: its loss often results in decreased pathogenicity, viability, or antibiotic resistance. In E. coli, ribosome stalling leads to collisions, which recruit the endonuclease SmrB to cleave the mRNA between collided ribosomes. These ribosomes are then targeted by the rescue factor tmRNA. Several lines of evidence suggest E. coli employ a second pathway to split antibiotic- inhibited ribosomes into subunits; however, a ribosome-splitting factor has yet to be identified. The DExH-box RNA helicase HrpA decreases E. coli sensitivity to ribosome-targeting antibiotics, suggesting this protein has a ribosome-associated function in antibiotic resistance. In fact, HrpA has been shown to rescue ribosomes stalled on reporter mRNA and resolve global antibiotic-induced ribosome collisions, indicating that it plays a role in clearing stacked ribosomes during translational stress. The proposed experiments will explore whether HrpA is a novel ribosome rescue factor that splits stalled ribosomes. Because ribosome collisions are critical for ribosome rescue in yeast, mammalian cells, and E. coli, Aim 1 will determine if ribosome collisions also recruit HrpA. Protein readout from a reporter-based assay in wild-type and ΔhrpA cells will be used to test if HrpA rescues collided ribosomes from mRNA. To determine if HrpA preferentially associates with collided ribosomes in vivo, sucrose density gradients will be used to determine if HrpA sediments with ribosome subunits, single ribosomes, or collided polysomes from cell lysates that are treated with ribosome-stalling antibiotics. Finally, the impact of HrpA on ribosome position and ribosome collisions transcriptome-wide will be explored using ribosome profiling in antibiotic-treated wild-type and ΔhrpA cells. Two S. cerevisiae RNA helicases in the same DExH-box family as HrpA are involved in ribosome splitting; therefore, Aim 2 will determine if HrpA similarly splits stalled ribosomes in E. coli. Because the protein Hsp15 preferentially associates with 50S ribosomal subunits from prematurely split ribosomes, HrpA splitting activity will be assayed in vivo by examining Hsp15 sedimentation in sucrose density gradients in antibiotic-treated wild-type and ΔhrpA cells. Finally, HrpA will be tested for its ability to split stalled ribosomes into subunits using an in vitro biochemical assay with purified components analyzed in sucrose density gradients. This assay will be used to determine ATPase activity and precise substrate recognition of HrpA. Characterization of HrpA will provide valuable insight into how E. coli mitigate the effects of ribosome-targeting antibiotics, marking it as a promising potential target of antimicrobial compounds in the treatment of human disease.