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

 描述(申请人提供)：在美国，每年有超过23000人死于抗生素耐药感染(CDC 2014)。为了对抗对常规抗生素日益增长的耐药性传播，开发新的抗生素将是必不可少的。通过与mRNAs碱基配对发挥作用的小非编码RNA(SRNAs)对基因的调控是此类抗生素的理想靶点，因为sRNAs控制着包括肠杆菌科细菌和革兰氏阳性病原体在内的多种细菌的细胞生长、适应性和毒力过程。长期目标是通过了解sRNA介导的基因调控的机制和蛋白质来识别新的抗生素靶点。初步结果表明，蛋白质RNase PH在调节sRNAs方面具有以前未意识到的作用。这项研究计划的目的是以大肠杆菌为模型系统，确定RNase PH与sRNA相互作用以保护它们免受降解的机制。本项目将检验这一假设，即RNase PH通过与SRNA 3‘末端区域相互作用来阻止其他RNase的结合和降解，从而选择性地调节SRNA降解。目的我将验证RNase PH通过阻止竞争RNase的结合来保护游离和mRNA结合的sRNAs免于降解的假设。核糖核酸酶PH对游离和mRNA结合的sRNA稳定性的影响将被检测。然后将测试RNase PH防止特定RNase降解SRNA的能力，并将检查其对SRNA丰度和细胞适合性的全球影响。AIM II将检验RNase PH结合但不降解其保护的sRNA的3‘末端的Rho非依赖性终止子的假设。直接与RNase PH结合的sRNA将被分离和测序，以评估它们的完整性。不同的SRNA功能对RNase PH稳定的贡献将通过嵌合SRNA构建和稳定性分析来检验，RNase PH的SRNA结合残基将通过突变、遗传筛选和稳定性分析来鉴定。在其结论中，该项目预期(目标一)展示RNase PH稳定sRNA的范围和作用，以及(目标II)提供对使这种保护发生的rRNA和RNase PH的特征的机械理解。了解RNase PH的这一新作用将为未来有效对抗sRNA介导的基因调控的新疗法的发展提供信息。

 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.