INITIATION OF MRNA DECAY IN BACILLUS SUBTILIS

枯草芽孢杆菌中 mRNA 衰变的启动

• 批准号: 3308250
• 负责人: DAVID H BECHHOFER
• 金额: $ 18.39万
• 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/3308250
• 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.

该建议的目的是了解信使RNA如何降解 始于革兰氏阳性细菌枯草菌。 迅速的 细菌mRNA的营业额是调节基因的重要元素 表达并允许微生物快速适应变化 环境条件。 从有关mRNA衰减的最新报告中 大肠杆菌以及我们与B. uttilis的合作，一般 可以提出启动mRNA衰变的模型。 简而言之 大多数mRNA分子衰减中的速率确定步骤是结合 消息的5'末端或附近的核酸酶。 核酸内切酶 然后在内部站点下游迁移并切割以启动 总体衰变。 该实验室的过去研究集中在mRNA的衰减上 由红霉素抗性基因ERMC编码。 ERMC的稳定 mRNA是通过核糖体在mRNA 5'端附近停滞不前而实现的 防止核糖核酸酶结合。 我们在ERMC mRNA衰减方面的工作使我们 提出以下方法来研究B中mRNA衰变的启动。 枯草厂： 1。与ERMC mRNA的5'末端结合并启动衰减的核酸酶 将通过使用设计的基材来表征 检测5'结合的核酸酶活性。 5'结合核酸酶将是 从枯草芽孢杆菌提取物纯化，N末端氨基酸序列 将确定，并使用寡核苷酸探针来识别 一个包含编码此核糖核酸酶的基因的克隆。 2。我们已经证明mRNA衰减可以从内部站点启动 被“ BS-RNase III”裂解，这是一种类似于大肠杆菌的核糖核酸酶 iii。 我们计划分离这种核酸酶并克隆编码的基因 它是为了评估其在RNA处理中的作用。 3。发夹结构在B的5'末端的稳定作用。 将确定枯草脂消息。 RNase e-like的存在 枯草芽孢杆菌中的活性将通过两种不同的方法进行测试。 一个 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。