Initiation of mRNA decay in Bacillus subtilis

描述(申请人提供)：项目摘要：我们实验室试图了解革兰氏阳性细菌枯草芽孢杆菌中信使RNA衰变的控制。虽然人们对信使核糖核酸合成(转录)和将信使核糖核酸翻译成蛋白质的机制和调控知道得很多，但对基因表达的中间步骤--信使核糖核酸的降解知之甚少。该方案中的实验集中在信使核糖核酸衰变的两个方面：决定其半衰期的信使核糖核酸酶的元件(例如，序列、结构)，以及启动和完成信使核糖核酸酶衰变所需的核糖核酸酶活性。缺乏1种或1种以上3‘到5’外切核糖核酸酶的枯草杆菌菌株的出现，将使研究单个核糖核酸酶在mRNA转换中的作用成为可能。本文将对3个小分子mRNAs的衰变过程进行详细的分析，为进一步研究小分子mRNAs的衰变过程奠定基础。建议进行实验以澄清：1)衰变的起始位置是什么？2)哪种核糖核酸酶(S)参与了衰变的起始？3)衰变机制如何处理稳定的二级结构？4)各种3‘到5’外切核糖核酸酶是如何结合和降解mRNA的？已知的4种枯草杆菌3‘到5’外切核酸酶--PNPase、RNase R、RNase PH和YhaM--的作用将在模型mRNAs的周转以及新发现的稳定在PNPase缺失突变株中的mRNAs中进行评估。将评估两种枯草杆菌内切核酸酶--RNaseJ1和RNaseJ2--在mRNA衰变中的可能参与。将建立一个体外系统，这将有助于探索纯化的核糖核酸酶的特性，这些核糖核酸酶将从大肠杆菌中过度表达和分离。相关性：信使核糖核酸(信使核糖核酸)是蛋白质合成的模板分子。细菌依靠快速的mRNA降解来适应不断变化的环境，这一过程的细节将在模式微生物枯草芽孢杆菌中进行详细研究。阐明这种细菌中的信使核糖核酸的机制可能会导致设计新的抗生素来抑制信使核糖核酸的衰退过程，从而防止成功的细菌在人体组织中的定植。

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.