Initiation of mRNA decay in Bacillus subtilis

枯草芽孢杆菌中 mRNA 降解的启动

• 批准号: 7921228
• 负责人: DAVID H BECHHOFER
• 金额: $ 3.67万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7921228
• 关键词: 5' -exoribonuclease; Address; Affect; Antibiotics; Bacillus subtilis; Bacteria; Binding; Binding Proteins; Characteristics; Elements; Endoribonucleases; Environment; Escherichia coli; Gene Expression; Gene Expression Profile; Genetic Transcription; Goals; Gram-Positive Bacteria; Half-Life; In Vitro; Individual; Kinetics; Laboratories; Laboratory Research; Lead; Messenger RNA; Modeling; Names; Organism; Phosphodiesterase I; Plasmids; Play; Polyribonucleotide Nucleotidyltransferase; Process; Proteins; RNA; RNA Decay; RNA chemical synthesis; Regulation; Ribonucleases; Role; Site; Small RNA; Structure; Study models; System; Translations; Work; design; endonuclease; endoribonuclease; human tissue; in vitro activity; mutant; overexpression; prevent; research study; ribonuclease E; ribonuclease PH; ribonuclease R; spleen exonuclease; stem

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.

描述（由申请人提供）： 项目摘要：我们的实验室致力于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 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 降解却知之甚少。该提案中的实验重点关注 mRNA 衰变的两个方面：决定其半衰期的 mRNA 元素（例如序列、结构），以及启动和完成 mRNA 衰变所需的核糖核酸酶活性。缺乏 1 种或多种 3'-5' 核糖核酸外切酶的枯草芽孢杆菌菌株的可用性将使检查单个核糖核酸酶在 mRNA 周转中的作用成为可能。 3种小mRNA的衰变特性已得到一定程度的研究，本文将对其进行详细分析，并将其作为一般mRNA衰变研究的模型。建议通过实验来澄清：1）衰变的起始位点是什么？ 2) 哪种核糖核酸酶参与腐烂的起始？ 3）衰变机制如何处理稳定的二级结构？ 4) 各种 3'-to-5' 核糖核酸外切酶如何结合和降解 mRNA？ 4 种已知的枯草芽孢杆菌 3' 至 5' 核糖核酸外切酶（PNPase、RNase R、RNase PH 和 YhaM）的作用将在模型 mRNA 以及新鉴定的稳定 mRNA 的周转中进行评估在 PNPase 缺陷突变株中。将评估 2 种枯草芽孢杆菌内切核糖核酸酶（RNase J1 和 RNase J2）在 mRNA 衰减中的可能参与。将建立一个体外系统，该系统可用于探测纯化核糖核酸酶的特性，该核糖核酸酶将从大肠杆菌中过表达并分离。相关性：信使 RNA (mRNA) 是合成蛋白质的模板分子。细菌依靠 mRNA 的快速衰减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。阐明这种细菌中 mRNA 的机制可能有助于设计新的抗生素，抑制 mRNA 的衰变过程，从而防止细菌在人体组织中成功定殖。