YloC, a new ribonuclease of Bacillus subtilis

YloC，枯草芽孢杆菌的新型核糖核酸酶

基本信息

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
项目类别：
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&apos -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

项目摘要

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研究了 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鉴定了几种核糖核酸酶（RNase）。枯草芽孢杆菌并阐明了它们在 mRNA 周转中所起的作用。缺乏枯草芽孢杆菌菌株的活力。所有已知的 3' 至 5' 核糖核酸外切酶促使我们继续鉴定其他 RNase 活性。利用经典的蛋白质生物化学，我们最近鉴定了一种新型 RNase，命名为 YloC。具有六聚体结构的核糖核酸内切酶，这是一种不寻常的特征，仅与另一种 RNase 共有： SARS-CoV家族的Nsp15蛋白初步实验表明，虽然YloC具有核糖核酸酶。尽管蛋白质在体外具有显着的活性，但它可能作为体内 RNA 相互作用的接头。与 YloC 的同源性在细菌物种中广泛存在，目前还没有任何关于其结构的公开信息。该蛋白质家族的成员。当前的提案旨在阐明 YloC 的结构和功能。如下： • 对 YloC 高度保守的残基进行诱变，以确定对多种特性的影响，包括核糖核酸酶活性、RNA 结合和结构，并阐明蛋白质的功能域。 • 通过随机序列SELEX 程序鉴定YloC 的高亲和力RNA 配体寡核苷酸和基因组RNA序列。 • 描述 YloC 与大肠杆菌多核苷酸磷酸化酶 (PNPase) 的强相互作用如何在大肠杆菌和可能的枯草芽孢杆菌中发挥小 RNA (sRNA) 调节作用。 • 确定与结合蛋白高度同源的大肠杆菌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。这项工作将建立在 YicC 结构初步确定的基础上。相关性：核糖核酸酶在 RNA 周转和加工中发挥着重要作用。与 RNA 分子结合并以酶促作用的蛋白质的研究将使抗菌剂的设计成为可能破坏这些蛋白质，从而干扰细菌细胞的生长。