Role of RNase PH in regulating sRNA stability and function in Escherichia coli

RNase PH 在调节大肠杆菌 sRNA 稳定性和功能中的作用

• 批准号: 9208055
• 负责人: Todd A Cameron
• 金额: $ 6.37万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-16 至 2018-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9208055
• 关键词: 5' -exoribonuclease; Address; Affect; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Base Pairing; Binding; Biological Assay; Biological Models; Cells; Centers for Disease Control and Prevention (U.S.); Co-Immunoprecipitations; Data; Development; Enterobacteriaceae; Environment; Escherichia coli; Future; Gene Expression Regulation; Genetic; Genetic Screening; Goals; Hybrids; Infection; Knowledge; Mediating; Medical; Messenger RNA; Molecular Chaperones; Monitor; Mutagenesis; Outcome; Patients; Pharmacologic Substance; Positioning Attribute; Process; Proteins; RNA Binding; RNA Biochemistry; RNA Decay; RNA Degradation; RNA Stability; Regulation; Research; Research Proposals; Resistance; Ribonucleases; Role; Structure; Testing; Therapeutic; Transfer RNA; Treatment Efficacy; Untranslated RNA; Virulence; Work; antimicrobial; bacterial resistance; cell growth; combat; cost; experience; fitness; global health; improved; inhibitor/antagonist; new therapeutic target; novel; novel therapeutics; pathogen; pathogenic bacteria; prevent; public health relevance; rho

 DESCRIPTION (provided by applicant): Each year in the U.S., more than 23 000 people die as a result of antibiotic resistant infections (CDC 2014). To combat the growing spread of resistances to conventional antibiotics, new antibiotic development will be essential. Gene regulation by small non-coding RNAs (sRNAs) that act by base-pairing with mRNAs is an ideal target for such antibiotics, since sRNAs control cell growth, adaptability, and virulence processes in a wide variety of bacteria, including Enterobacteriaceae and Gram-positive pathogens. The long-term goal is to identify new antibiotic targets by understanding the mechanisms and proteins involved in sRNA-mediated gene regulation. Preliminary results indicate that the protein RNase PH has a previously unrealized role in regulating sRNAs. The objective of this research proposal is to determine the mechanisms by which RNase PH interacts with sRNAs to protect them from degradation, using Escherichia coli as a model system. This project will test the hypothesis that RNase PH selectively regulates sRNA degradation by interacting with sRNA 3' terminal regions to block binding and degradation by other RNases. Aim I will test the hypothesis that RNase PH protects both free and mRNA-bound sRNAs from degradation by preventing the binding of competing RNases. The effect of RNase PH on the stability of free and mRNA-bound sRNAs will be examined. Then the ability of RNase PH to protect against sRNA degradation by specific RNases will be tested, and its global impact on sRNA abundance and cellular fitness will also be examined. Aim II will test the hypothesis that RNase PH binds, but does not degrade, the Rho-independent terminators at the 3' termini of the sRNAs it protects. The sRNAs that directly bind to RNase PH will be isolated and sequenced to assess their integrity. The contributions of different sRNA features to stabilization by RNase PH will be examined through chimeric sRNA constructs and stability assays, and the sRNA-binding residues of RNase PH will be identified through mutagenesis, genetic screening, and stability assays. At its conclusion, this project is expected to (Aim I) demonstrate the scope and role of sRNA stabilization by RNase PH, as well as (Aim II) provide a mechanistic understanding of the features of sRNAs and RNase PH that enable this protection to occur. Understanding this novel role of RNase PH will inform the future development of new therapeutics effective against sRNA- mediated gene regulation.

 描述（由申请人提供）：每年在美国，超过23000人死于抗生素耐药性感染（CDC 2014）。为了对抗传统抗生素耐药性的日益蔓延，新抗生素的开发将是必不可少的。通过与mRNA碱基配对起作用的小的非编码RNA（sRNA）的基因调控是此类抗生素的理想靶标，因为sRNA控制多种细菌（包括肠杆菌科和革兰氏阳性病原体）中的细胞生长、适应性和毒力过程。长期目标是通过了解参与sRNA介导的基因调控的机制和蛋白质来确定新的抗生素靶点。初步结果表明，蛋白质RNase PH在调节sRNA中具有以前未实现的作用。本研究的目的是确定RNase PH与sRNA相互作用以保护它们免受降解的机制，使用大肠杆菌作为模型系统。该项目将测试RNase PH通过与sRNA 3'末端区域相互作用以阻断其他RNase的结合和降解来选择性地调节sRNA降解的假设。目的验证RNase PH通过阻止竞争性RNase的结合来保护游离和与mRNA结合的sRNA不被降解的假设。将检查RNase PH对游离和mRNA结合的sRNA的稳定性的影响。然后将测试RNase PH保护免受特定RNase降解的能力，并检查其对sRNA丰度和细胞适应性的整体影响。目的II将检验RNase PH结合但不降解其保护的sRNA的3'末端处的Rho非依赖性终止子的假设。将直接结合RNase PH的sRNA分离并测序，以评估其完整性。将通过嵌合sRNA构建体和稳定性试验检查不同sRNA特征对RNase PH稳定性的贡献，并通过诱变、遗传筛选和稳定性试验鉴定RNase PH的sRNA结合残基。在其结论中，该项目预计（目的I）证明RNA酶PH稳定sRNA的范围和作用，以及（目的II）提供对使这种保护发生的sRNA和RNA酶PH特征的机制理解。了解RNase PH的这种新作用将为未来开发有效对抗sRNA介导的基因调控的新疗法提供信息。