YloC, a new ribonuclease of Bacillus subtilis

项目摘要：多年来，我们实验室一直在研究mrna衰变的基本过程。 革兰氏阳性菌的典范，枯草芽孢杆菌。我们已经鉴定了几种核糖核酸酶 并阐明了它们在mRNA周转中所起的作用。一株缺乏活力的枯草杆菌菌株 所有已知的3‘至5’外切核糖核酸酶促使我们寻找更多的核糖核酸酶活性。 利用经典的蛋白质生物化学，我们最近发现了一种新的核糖核酸酶，命名为YloC。YloC是一位 具有六聚体结构的内切核糖核酸酶，这是一种不寻常的特征，只与另一种核糖核酸酶相同： SARS冠状病毒家族的Nsp15蛋白。初步实验表明，尽管YloC具有核糖核酸酶 在体外的活性，它可能作为体内RNA相互作用的适配器。尽管具有重要意义的蛋白质 YloC的同源性在细菌物种中广泛存在，目前还没有关于任何YloC的结构的公开信息 这个蛋白质家族的成员。目前的建议旨在澄清YloC的结构和功能，因为 以下是： ·诱变YloC高度保守的残基，以确定对几个性质的影响--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。 ·利用随机序列通过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 在大肠杆菌和枯草杆菌中可能参与小RNA(SRNA)的调节。 ·确定与An结合的高度同源的E.ColiYicC蛋白的三维结构 RNA底物，以及来自嗜热细菌物种的YloC和/或其同系物的结构。 这项工作将建立在对YicC结构的初步确定的基础上。 相关性：核糖核酸酶在RNA周转和加工中起着至关重要的作用。透彻了解 结合在RNA分子上并在其上进行酶作用的蛋白质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。

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.