INITIATION OF MRNA DECAY IN BACILLUS SUBTILIS

枯草芽孢杆菌中 mRNA 衰变的启动

• 批准号: 2186314
• 负责人: DAVID H BECHHOFER
• 金额: $ 20.1万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186314
• 关键词: Bacillus subtilis; bacterial genetics; chemical cleavage; enzyme activity; messenger RNA; molecular cloning; nucleic acid probes; nucleic acid sequence; protein degradation; protein sequence; ribonuclease III; southern blotting; temperature sensitive mutant

The goal of this proposal is to understand how messenger RNA degradation is initiated in the Gram-positive bacterium Bacillus subtilis. Rapid turnover of bacterial mRNA is an important element in regulating gene expression and allows quick adaption of microorganisms to changing environmental conditions. From recent reports concerning mRNA decay in Escherichia coli, as well as our own work with B. subtilis, a general model for the initiation of mRNA decay can be proposed. Briefly, the rate-determining step in decay of most mRNA molecules is the binding of an endonuclease at or near the 5' end of the message. The endonuclease then migrates downstream and cleaves at an internal site to initiate overall decay. Past research in this laboratory has concentrated on the decay of mRNA encoded by the erythromycin resistance gene ermC. Stabilization of ermC mRNA is achieved by ribosome stalling near the mRNA 5' end, which prevents ribonuclease binding. Our work on ermC mRNA decay has led us to propose the following ways to study initiation of mRNA decay in B. subtilis: 1. The nuclease that binds to the 5' end of ermC mRNA and initiates decay will be characterized through the use of substrates that are designed to detect a 5'-binding nuclease activity. The 5'-binding nuclease will be purified from B. subtilis extracts, an N-terminal amino acid sequence will be determined, and oligonucleotide probes will be used to identify a clone containing the gene encoding this ribonuclease. 2. We have shown that mRNA decay can initiate from an internal site that is cleaved by "Bs-RNase III", a ribonuclease that resembles E. coli RNase III. We plan to isolate this nuclease and clone the gene that encodes it in order to assess its role in RNA processing. 3. The stabilizing effect of a hairpin structure at the 5' end of a B. subtilis message will be determined. The presence of an RNase E-like activity in B. subtilis will be tested by two different methods. A genetic technique to select for DNA fragments that encode endonuclease cleavage sites is proposed.

这项计划的目标是了解信使RNA降解 在革兰氏阳性细菌枯草芽孢杆菌中起始。 快速 细菌mRNA的周转是基因调控的重要元件 表达，并允许微生物快速适应变化 环境条件 从最近有关mRNA衰变的报道来看， 大肠杆菌，以及我们自己的工作与B。subtilis，a general 可以提出mRNA衰变起始的模型。 采用离体垂体碎片 大多数mRNA分子衰变的速率决定步骤是结合 位于或靠近信使5'端的核酸内切酶。 核酸内切酶 然后向下游迁移并在内部部位裂开，开始 整体衰减 该实验室过去的研究主要集中在mRNA的衰变 由红霉素抗性基因ermC编码。 ermC的稳定化 mRNA通过核糖体在mRNA 5'端附近停滞而获得， 阻止核糖核酸酶结合。 我们对ermC mRNA衰变的研究使我们 提出以下方法来研究B中mRNA衰变的起始。 枯草杆菌： 1.与ermC mRNA的5'末端结合并启动衰变的核酸酶 将通过使用被设计成 检测5’-结合核酸酶活性。 5 '-结合核酸酶将是 从B纯化。枯草杆菌提取物，N-末端氨基酸序列 将确定，并将使用寡核苷酸探针来鉴定 含有编码这种核糖核酸酶的基因的克隆。 2.我们已经证明，mRNA的衰变可以从一个内部位点开始， 被“Bs-RNase III”（一种类似于E.杆菌rna酶 三. 我们计划分离这种核酸酶并克隆编码 为了评估它在RNA加工中的作用。 3.在B的5'末端的发夹结构的稳定作用。 将确定subtilis消息。 RNA酶E样的存在 B中的活性。将通过两种不同的方法检测枯草芽孢杆菌。 一 选择编码核酸内切酶的DNA片段的遗传技术 提出了切割位点。