Initiation of mRNA decay in Bacillus subtilis

描述（由申请人提供）：项目摘要：我们的实验室旨在了解革兰氏阳性细菌枯草芽孢杆菌中信使RNA衰变的控制。虽然对mRNA合成（转录）和mRNA翻译成蛋白质的机制和调控了解很多，但对基因表达的中间步骤-mRNA降解知之甚少。该提案中的实验集中在mRNA衰变的两个方面：元素（例如，序列、结构），其决定mRNA的半衰期，以及mRNA衰变的起始和完成所需的核糖核酸酶活性。B的可用性。缺乏一种或多种3 ′-至5 ′核糖核酸外切酶的枯草杆菌菌株将使得有可能检查单个核糖核酸酶在mRNA周转中的作用。3个小mRNA的衰变，其特征已在一定程度上进行了研究，将详细分析，并将作为模型的mRNA衰变的研究一般。提出实验来阐明：1）衰变的起始点是什么？2)哪种核糖核酸酶参与衰变的起始？3)衰变机制如何处理稳定的二级结构？以及4）各种3 '至5'核糖核酸外切酶如何结合和降解mRNA？4已知的B的作用。将在模型mRNA以及在PNPase-deficient突变株中稳定的新鉴定的mRNA的周转中评估枯草杆菌3 '至5'核糖核酸外切酶-PNPaseR、RNasePH和YhaM。2 B的可能参与。将评估枯草杆菌核糖核酸内切酶--RNA酶J1和RNA酶J2 --在mRNA衰变中的作用。将建立一个体外系统，这将是有用的，在探测纯化的核糖核酸酶，这将是过表达和分离从大肠杆菌的特性。杆菌相关性：信使RNA（mRNA）是蛋白质合成的模板分子。细菌依靠快速的mRNA衰变来适应不断变化的环境，这一过程的细节将在模型微生物枯草芽孢杆菌中详细研究。阐明这种细菌中mRNA的机制可能会导致设计新的抗生素，抑制mRNA的衰变过程，从而防止细菌在人体组织中成功定植。

DESCRIPTION (provided by applicant): Project Summary: Our laboratory seeks to understand the control of messenger RNA decay in the gram-positive bacterium, Bacillus subtilis. While much is known about the mechanism and regulation of mRNA synthesis (transcription) and translation of mRNA into protein, little is known about the intermediate step in gene expression-degradation of mRNA. Experiments in this proposal focus on 2 aspects of mRNA decay: the elements (e.g., sequences, structures) of an mRNA that determine its half-life, and the ribonuclease activities that are required for initiation and completion of mRNA decay. The availability of B. subtilis strains that are deficient in 1 or more 3'-to-5' exoribonucleases will make it possible to examine the role of individual ribonucleases in mRNA turnover. The decay of 3 small mRNAs, whose characteristics have been studied to some extent, will be analyzed in detail and will serve as models for the study of mRNA decay generally. Experiments are proposed to clarify: 1) what is the initiation site for decay? 2) which ribonuclease(s) participates in initiation of decay? 3) how does the decay mechanism deal with stable secondary structure? and 4) how do the various 3'-to-5' exoribonucleases bind and degrade mRNA? The role of the 4 known B. subtilis 3'-to-5' exoribonucleases -- PNPase, RNase R, RNase PH, and YhaM-will be assessed in the turnover of model mRNAs, as well as newly-identified mRNAs that are stabilized in a PNPase-deficient mutant strain. The likely participation of 2 B. subtilis endoribonucleases -- RNase J1 and RNase J2 -in mRNA decay will be assessed. An in vitro system will be established that will be useful in probing the characteristics of purified ribonucleases, which will be overexpressed and isolated from E. coli. Relevance: Messenger RNA (mRNA) is the template molecule upon which proteins are synthesized. Bacteria rely on rapid mRNA decay to adapt to changing environments, and the details of this process will be studied in detail in the model microorgansim, Bacillus subtilis. Elucidating the mechanism of mRNA in this bacterium could lead to the design of new antibiotics that inhibit the mRNA decay process and thereby prevent successful bacterial colonization of human tissues.