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

HrpA 在大肠杆菌核糖体相关质量控制中的作用

• 批准号: 10738264
• 负责人: Annabelle Campbell
• 金额: $ 4.13万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10738264
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Binding; Biochemical; Biological Assay; Borrelia burgdorferi; Cells; Cues; Dependence; Development; Escherichia coli; Escherichia coli Proteins; Family; Future; Growth; Human; Hydrolysis; Hypersensitivity; In Vitro; Lyme Disease; Mammalian Cell; Messenger RNA; Modification; Mutation Analysis; Orthologous Gene; Pathogenicity; Pathway interactions; Peptides; Pharmaceutical Preparations; Polyribosomes; Positioning Attribute; Predisposition; Process; Proteins; Proteobacteria; Quality Control; RNA; RNA Helicase; Recycling; Reporter; Resources; Ribosomes; Role; Saccharomyces cerevisiae; Sedimentation process; Site; Stress; Sucrose; System; Techniques; Testing; Translations; Yeasts; antimicrobial; density; endonuclease; experimental study; fitness; genome-wide; human disease; in vivo; insight; mouse model; novel; pathogenic bacteria; peptidyl-tRNA; premature; recruit; ribosome profiling; tmRNA; transcriptome; transcriptome sequencing; translation factor; transmission process

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.

项目总结/摘要 延长核糖体经常遇到障碍，导致核糖体停滞。停滞核糖体 然后被拯救因子靶向回收核糖体亚基，并靶向有缺陷的mRNA和新生 衰变肽这一过程对于细菌的细胞适应性至关重要：它的丧失通常会导致细胞生长能力的降低。 致病性、生存能力或抗生素耐药性。在大肠在大肠杆菌中，核糖体停滞导致碰撞， 核酸内切酶SmrB切割碰撞的核糖体之间的mRNA。然后这些核糖体被 拯救因子tmRNA。几条证据表明E.大肠杆菌使用第二种途径来分解抗生素， 抑制核糖体分解成亚基;然而，核糖体分裂因子尚未被鉴定。DExH盒 RNA解旋酶HrpA降低E.大肠杆菌对核糖体靶向抗生素的敏感性，表明这种蛋白质具有 核糖体在抗生素耐药性中的相关功能。事实上，HrpA已经被证明可以拯救停滞的核糖体， 对报告基因mRNA和解决全球性免疫诱导的核糖体碰撞，表明它在 在翻译应激期间清除堆积的核糖体。拟议的实验将探讨HrpA是否是 一种能分裂停滞核糖体的新型核糖体拯救因子。因为核糖体碰撞对于核糖体 酵母、哺乳动物细胞和E. coli中，Aim 1将确定核糖体碰撞是否也募集HrpA。 将使用野生型和ΔhrpA细胞中基于荧光素酶的测定的蛋白质读数来测试HrpA是否拯救 核糖体与mRNA的碰撞。为了确定HrpA在体内是否优先与碰撞的核糖体结合， 蔗糖密度梯度将用于确定HrpA是否与核糖体亚单位，单个核糖体， 或者来自用核糖体停滞抗生素处理的细胞裂解物的碰撞的多核糖体。最后，影响 将使用核糖体分析探索核糖体位置上的hrpA和转录组范围内的核糖体碰撞 在抗肿瘤处理的野生型和ΔhrpA细胞中。两个S同一DExH盒家族中的酿酒酵母RNA解旋酶 由于HrpA参与核糖体分裂;因此，目标2将确定HrpA是否类似地停止分裂 E.杆菌由于Hsp 15蛋白优先与来自大肠杆菌的50 S核糖体亚基结合， 在核糖体过早分裂的情况下，HrpA分裂活性将通过检查Hsp 15在细胞中的沉降来体内测定。 蔗糖密度梯度在拟南芥处理的野生型和ΔhrpA细胞。最后，将测试HrpA的能力， 使用体外生物化学测定将停滞的核糖体分裂成亚基， 蔗糖密度梯度该试验将用于测定ATP酶活性和精确底物 识别HrpA。HrpA的特性将提供有价值的洞察如何E。大肠杆菌可以减轻 核糖体靶向抗生素，标志着它作为一个有前途的潜在目标的抗菌化合物在 治疗人类疾病。