Initiation of mRNA decay in Bacillus subtilis

项目概述：本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。虽然人们对mRNA合成（转录）和mRNA转化为蛋白质的机制和调控了解甚多，但对基因表达的中间步骤- mRNA降解知之甚少。本实验主要关注mRNA衰变的两个方面：决定mRNA半衰期的元素（如序列、结构），以及mRNA衰变开始和完成所需的核糖核酸酶活性。枯草芽孢杆菌菌株缺乏1个或多个3‘至5’外核糖核酸酶，这将使研究单个核糖核酸酶在mRNA周转中的作用成为可能。我们将详细分析3种小mRNA的衰变，它们的特性已经得到了一定程度的研究，并将作为研究mRNA衰变的一般模型。提出了实验来澄清：1)什么是衰变的起始点？2)哪种核糖核酸酶参与了衰变的开始？3)衰变机制如何处理稳定的二级结构？4)各种3‘到5’的外核糖核酸酶是如何结合和降解mRNA的？将评估4种已知枯草芽孢杆菌3‘至5’外核糖核酸酶（PNPase, RNase R， RNase PH和yham）在模型mrna周转中的作用，以及在PNPase缺陷突变株中稳定的新鉴定的mrna。将评估2种枯草芽孢杆菌核糖核酸内切酶（RNase J1和RNase J2）参与mRNA衰变的可能性。将建立一个体外系统，用于探测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表达和分离。相关性：信使RNA （mRNA）是合成蛋白质的模板分子。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进行详细研究。阐明mRNA在这种细菌中的作用机制可能会导致设计新的抗生素来抑制mRNA的衰变过程，从而阻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.