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

基本信息

批准号：
10537191
负责人：
Annabelle Campbell
金额：
$ 4.04万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
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/10537191
关键词：
ATP phosphohydrolase Affect Antibiotic Resistance Antibiotic Therapy Antibiotics Bacteria Binding Biochemical Biological Assay Borrelia burgdorferi Cells Dependence Development Escherichia coli Escherichia coli Proteins Family Future Growth Human Hydrolysis Hypersensitivity In Vitro Lyme Disease Mammalian Cell Messenger RNA Modification Mus Mutation Analysis Orthologous Gene Pathogenicity Pathway interactions Peptides Pharmaceutical Preparations Play Polyribosomes Positioning Attribute Predisposition Process Proteins Proteobacteria Quality Control RNA RNA Helicase Reporter Resources Ribosome Inactivation Ribosomes Role Saccharomyces cerevisiae Sedimentation process Site Stress Sucrose System Techniques Testing Translations Yeasts antimicrobial base density endonuclease experimental study fitness genome-wide human disease in vivo insight novel pathogenic bacteria peptidyl-tRNA premature recruit ribosome profiling tmRNA transcriptome transcriptome sequencing transmission process

项目摘要

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.

项目概要/摘要延长核糖体经常遇到导致核糖体停滞的障碍。然后被救援因子靶向回收核糖体亚基并靶向有缺陷的 mRNA 和新生这个过程对于细菌的细胞适应性至关重要：它的损失通常会导致细菌的细胞适应性下降。在大肠杆菌中，核糖体停滞会导致碰撞，从而招募核酸内切酶 SmrB 在碰撞的核糖体之间切割 mRNA，然后这些核糖体被靶向。多种证据表明大肠杆菌采用第二种途径来分解抗生素- 抑制核糖体形成亚基；然而，核糖体分裂因子尚未确定。 RNA 解旋酶 HrpA 降低大肠杆菌对核糖体靶向抗生素的敏感性，表明该蛋白具有抗生素耐药性中的核糖体相关功能事实上，HrpA 已被证明可以挽救停滞的核糖体。报告基因 mRNA 并解决全局抗生素诱导的核糖体碰撞，表明它在所提出的实验将探索 HrpA 是否在翻译应激过程中清除堆积的核糖体。一种新颖的核糖体救援因子，可以分裂停滞的核糖体，因为核糖体碰撞对于核糖体至关重要。为了拯救酵母、哺乳动物细胞和大肠杆菌，目标 1 将确定核糖体碰撞是否也招募 HrpA。野生型和 ΔhrpA 细胞中基于报告基因的测定中的蛋白质读数将用于测试 HrpA 是否可以拯救为了确定 HrpA 是否优先与体内的碰撞核糖体结合，蔗糖密度梯度将用于确定 HrpA 是否与核糖体亚基、单个核糖体、或来自用核糖体停滞抗生素处理的细胞裂解物的碰撞多核糖体最后，影响。将使用核糖体分析来探索转录组范围内核糖体位置和核糖体碰撞的 HrpA 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。由于 HrpA 参与核糖体分裂；因此，目标 2 将确定 Hrp A 分裂是否停滞；大肠杆菌中的核糖体因为蛋白质 Hsp15 优先与来自大肠杆菌的 50S 核糖体亚基结合。过早分裂核糖体，HrpA 分裂活性将通过检查 Hsp15 沉降在体内进行测定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。使用体外生化测定将核糖体停滞为亚基，并在其中分析纯化成分该测定将用于测定 ATP 酶活性和精确底物。 HrpA 的识别将为大肠杆菌如何减轻影响提供有价值的见解。核糖体靶向抗生素，使其成为抗菌化合物的一个有前途的潜在靶点治疗人类疾病。