YloC, a new ribonuclease of Bacillus subtilis

YloC，枯草芽孢杆菌的新型核糖核酸酶

• 批准号: 10736779
• 负责人: DAVID H BECHHOFER
• 金额: $ 35.49万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736779
• 关键词: 5' -exoribonuclease; Adaptor Signaling Protein; Affect; Affinity; Alanine; Awareness; Bacillus subtilis; Bacteria; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Process; Biology; Biophysics; C-terminal; Catalysis; Cells; Characteristics; Code; Coronavirus; Endoribonucleases; Enzyme Inhibition; Enzymes; Escherichia coli; Exonuclease; Family; Family member; Gene Deletion; Genes; Gram-Positive Bacteria; Homologous Gene; In Vitro; Laboratories; Ligands; Messenger RNA; Modeling; Mutagens; N-terminal; Names; Nucleotides; Oligonucleotides; Organism; Outcome; Pancreatic ribonuclease; Physiological Processes; Play; Polyribonucleotide Nucleotidyltransferase; Procedures; Process; Property; Protein Biochemistry; Protein Family; Proteins; Publishing; RNA; RNA Binding; RNA Sequences; RNA chemical synthesis; Regulation; Regulatory Element; Reporting; Ribonucleases; Role; SARS coronavirus; Sequence Homology; Small RNA; Specific qualifier value; Specificity; Structure; Tertiary Protein Structure; Testing; Transcript; Virulence; Work; antibiotic design; antimicrobial drug; cell growth; design; endonuclease; environmental change; experimental study; follow-up; genome-wide; genomic RNA; in vitro activity; in vivo; insight; mRNA Decay; member; microorganism; model organism; mutant; novel; poly A specific exoribonuclease; prototype; recruit; response; thermophilic organism; three dimensional structure

PROJECT SUMMARY: Our laboratory has, for many years, studied the essential process of mRNA decay in the model Gram-positive bacterium, Bacillus subtilis. We have identified several ribonuclease (RNase) enzymes of B. subtilis and have elucidated the role they play in mRNA turnover. The viability of a B. subtilis strain lacking all of the known 3’-to-5’ exoribonucleases prompted us to pursue identification of additional RNase activities. Using classic protein biochemistry, we recently identified a novel RNase, named YloC. YloC is an endoribonuclease with a hexameric structure, an unusual characteristic that is shared with only one other RNase: the Nsp15 protein of the SARS-CoV family. Initial experiments suggest that, although YloC has ribonuclease activity in vitro, it may function as an adapter for RNA interactions in vivo. Although proteins with significant homology to YloC are widespread in bacterial species, there is no published information on the structure of any member of this protein family. The current proposal seeks to elucidate the structure and function of YloC, as follows: • Mutagenize highly conserved residues of YloC to determine the effect on several properties – including ribonuclease activity, RNA binding, and structure – and to clarify functional domains of the protein. • Identify high-affinity RNA ligands of YloC via SELEX procedures with random-sequence oligonucleotides and with genomic RNA sequences. • Characterize how the strong interaction of YloC with E. coli polynucleotide phosphorylase (PNPase) acts in small RNA (sRNA) regulation in E. coli and possibly in B. subtilis. • Determine the three-dimensional structure of the highly homologous E. coli YicC protein bound to an RNA substrate, as well as the structure of YloC and/or its homologs from thermophilic bacterial species. This work will build on an initial determination of the structure of YicC. RELEVANCE: Ribonucleases play essential roles in RNA turnover and processing. A thorough understanding of the proteins that bind to and act enzymatically on RNA molecules will enable design of antimicrobial agents that disrupt such proteins and thereby interfere with bacterial cell growth.

项目摘要：我们的实验室多年来一直在研究mRNA衰变的基本过程 模型革兰氏阳性细菌，枯草芽孢杆菌。我们已经确定了几种核糖核酸酶（RNase）酶 枯草芽孢杆菌，并阐明了它们在mRNA周转率中所起的作用。缺乏枯草芽孢杆菌菌株的生存能力 所有已知的3'至5'驱核核酸酶促使我们追求其他RNase活动的识别。 使用经典的蛋白质生物化学，我们最近确定了一种名为Yloc的新型RNase。 Yloc是一个 具有六聚体结构的内核酸酶，这是一种不寻常的特征，仅与另一个RNase共享： SARS-COV家族的NSP15蛋白。最初的实验表明，尽管YLOC的核糖核酸酶 在体外活性，它可能充当体内RNA相互作用的衔接子。虽然具有明显的蛋白质 与Yloc的同源性在细菌物种中是广泛的，没有关于任何的结构的发布信息 这个蛋白质家族的成员。当前的建议旨在阐明YLOC的结构和功能，作为 以下内容： •诱变高度组成YLOC的残差，以确定对几种特性的影响 - 包括 核糖核酸酶活性，RNA结合和结构 - 并阐明蛋白质的功能结构域。 •通过随机序列识别YLOC的高亲和力RNA配体 寡核苷酸和基因组RNA序列。 •表征YLOC与大肠杆菌多核苷酸磷酸化酶（PNPase）的强相互作用如何 作用于大肠杆菌中的小RNA（SRNA）调节，并在枯草芽孢杆菌中进行。 •确定高度同源大肠杆菌YICC蛋白的三维结构 RNA底物，以及来自嗜热细菌物种的YLOC和/或其同源物的结构。 这项工作将基于YICC结构的初始确定。 相关性：核糖核酸酶在RNA更新和处理中起着重要作用。彻底的理解 在RNA分子上结合并作用于酶促作用的蛋白质将实现抗菌剂的设计 破坏这种蛋白质，从而干扰细菌细胞生长。