Altering translation allows E. coli to overcome G-quadruplex stabilizers

G-quadruplex (G4) structures can form in guanine-rich DNA or RNA and have been found to modulate cellular processes including replication, transcription, and translation. Many studies on the cellular roles of G4s have focused on eukaryotic systems, with far fewer probing bacterial G4s. Using a chemical-genetic approach, we identified genes in Escherichia coli that are important for growth in G4-stabilizing conditions. Reducing levels of elongation factor Tu or slowing translation elongation with chloramphenicol suppress the effects of G4 stabilization. In contrast, reducing the expression of certain translation termination or ribosome recycling proteins is detrimental to growth in G4-stabilizing conditions. Proteomic and transcriptomic analyses demonstrate that ribosome assembly factors and other proteins involved in translation are less abundant in G4-stabilizing conditions. Our results suggest that RNA G4s can present barriers to E. coli growth and reducing the rate of translatio..., Strain construction All cells used in this study are derived from an Escherichia coli MG1655 parent strain unless otherwise specified. For CRISPR interference strains, strains were a gift from Jason Peters (46). To generate E. coli knockout strains, P1 transductions were carried out using Keio collection strains as the donor strain (63,64). P1 phage lysate was grown on Keio collection donor strains, which was used to transduce the MG1655 strains or CRISPR interference strains (to make the tolC knockout of selected CRISPRi strains) which were sensitive to kanamycin. To validate strains, transductions were grown on LB plates supplemented with 50 µg/mL kanamycin and screened using colony PCR to validate proper insertion of the kanamycin resistance cassette. To remove the kanamycin resistant cassette from MG1655 tolC::kan to enable additional P1 transductions in this strain, MG1655 tolC::kan electrocompetent cells were generated and transformed with a plasmid encoding the FLP recombinase (p..., , # Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes [https://doi.org/10.5061/dryad.zcrjdfnn9](https://doi.org/10.5061/dryad.zcrjdfnn9) ## Description of the data and file structure The data included in this Dryad submission was collected in order to understand how the model organism* Escherichia coli* overcomes stabilized G-quadruplexes. This work involved a multi-omics approach to studying how the G-quadruplex stabilizers NMM and Braco-19 impact growth, gene importance, and mRNA/proteomic abundance in G-quadruplex stabilizing conditions. ### Files and variables #### File: Western\_total\_protein\_stain\_1.tif **Description:** Total protein staining for western blots assessing EF-Tu abundance in ∆tolC, ∆tolC tufA::kan, and ∆tolC tufB::kan cells. Order of gel is ∆tolC, ∆tolC tufA::kan, and ∆tolC tufB::kan at three different dilutions added into the gel. This is the total protein stain image used in the figure of the manuscript. #### File: Wester...G-四链体（G-quadruplex, G4）可在富含鸟嘌呤的DNA或RNA中形成，并已被证实可调控包括复制、转录与翻译在内的多种细胞过程。目前针对G4细胞功能的大量研究多聚焦于真核生物系统，对细菌G4的探索则相对匮乏。本研究通过化学遗传学方法，在大肠杆菌（Escherichia coli）中筛选出在G4稳定化条件下对生长至关重要的基因。下调延伸因子Tu（elongation factor Tu, EF-Tu）的表达水平，或用氯霉素减缓翻译延伸速率，均可抑制G4稳定化带来的生长影响。与之相反，下调某些翻译终止蛋白或核糖体回收蛋白的表达，则会使大肠杆菌在G4稳定化条件下的生长受到损害。蛋白质组与转录组分析显示，在G4稳定化条件下，核糖体组装因子及其他参与翻译过程的蛋白丰度均有所降低。本研究结果表明，RNA G4可成为大肠杆菌生长的障碍，而降低翻译速率…… 菌株构建： 除非另有说明，本研究中使用的所有菌株均源自大肠杆菌MG1655亲本菌株。CRISPR干扰菌株由Jason Peters馈赠（46）。为构建大肠杆菌敲除菌株，本研究以Keio敲除文库（Keio collection）菌株作为供体菌株开展P1转导实验（63,64）：首先在Keio敲除文库供体菌株中制备P1噬菌体裂解液，再利用该裂解液转导MG1655菌株或CRISPR干扰菌株（以构建对应CRISPRi菌株的tolC敲除株，该菌株对卡那霉素敏感）。菌株验证阶段，将转导后的菌体接种于添加50 μg/mL卡那霉素的LB平板上，并通过菌落PCR验证卡那霉素抗性基因盒的正确插入。为从MG1655 tolC::kan菌株中移除卡那霉素抗性基因座以支持该菌株后续的P1转导实验，本研究制备了MG1655 tolC::kan电转化感受态细胞，并转化携带FLP重组酶（FLP recombinase）的质粒（p……）。 # 调控翻译可使大肠杆菌克服化学稳定化的G-四链体 [https://doi.org/10.5061/dryad.zcrjdfnn9] ## 数据与文件结构说明 本Dryad数据集收录的数据旨在解析模式生物大肠杆菌如何应对稳定化的G-四链体。本研究采用多组学策略，探究G-四链体稳定剂NMM与Braco-19在G4稳定化条件下对大肠杆菌生长、基因重要性以及mRNA/蛋白质组丰度的影响。 ### 文件与变量 #### 文件：Western_total_protein_stain_1.tif **描述**：用于蛋白质免疫印迹实验的总蛋白染色结果，用以检测ΔtolC、ΔtolC tufA::kan与ΔtolC tufB::kan菌株中EF-Tu的丰度。凝胶上样顺序依次为：三种不同稀释度的ΔtolC、ΔtolC tufA::kan与ΔtolC tufB::kan菌体样品。该图像即为论文配图中使用的总蛋白染色图。 #### 文件：Wester...