SWITCH GOVERNING BACILLUS MOTHER CELL GENE EXPRESSION

控制芽孢杆菌母细胞基因表达的开关

• 批准号: 2608912
• 负责人: LEE R KROOS
• 金额: $ 22.46万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2608912
• 关键词: Bacillus subtilis; DNA directed RNA polymerase; DNA footprinting; bacterial genetics; binding proteins; developmental genetics; endopeptidases; gene induction /repression; gene mutation; genetic promoter element; genetic transcription; immunologic assay /test; laboratory rabbit; microorganism reproduction; nucleic acid sequence; protein purification; protein structure; regulatory gene; spores; transcription factor

The long-term objective of this research is to understand how gene expression is controlled temporally and spatially during the relatively simple developmental process of Bacillus subtilis sporulation. This process involves a highly ordered program of gene expression and morphological change, and provides an attractive experimental system for elucidating fundamental mechanisms of gene regulation. These studies ar of broad significance and will have important implications for public health, because gene expression is temporally and spatially regulated during the cell cycle, as well as during adaptive and developmental responses, including during the interaction of a pathogen with its host. The proposed research focuses on three key regulators that comprise a switch governing the transition from early to late gene expression in th mother-cell compartment of the developing sporangium. The switch is initiated in response to a signal from the other compartment of the sporangium, the forespore. This step involves processing of an inactive precursor, pro-sigmaK, to sigmaK, a subunit of RNA polymerase that directs late gene transcription in the mother cell. The SpoIVFB protein may be the protease that processes pro-sigmaK, or a regulator of the processing reaction. To elucidate the mechanism of processing, an antibody probe for SpoIVFB will be developed and used to determine its intracellular location, as well as to isolate fractions enriched for SpoIVFB, which will be used to reconstitute pro-sigmaK processing in vitro. Mutations that bypass the need for SpoIVFB in sporulation will be characterized to determine whether SpoIVFB is likely to be the pro- sigmaK processing enzyme and, if not, to identify the protease. The appearance of sigmaK somehow causes a decrease in the level of sigmaE, which directs early mother-cell gene transcription. This halts transcripts of spoIIID, which encodes the second key component of the switch. The SpoIIID protein activates or represses transcription of bot early and late mother-cell genes. Mutations in sigK (encoding sigmaK) will be constructed and used to determine whether sigmaK must be transcriptionally active to negatively regulate the sigmaE and SpoIIID levels. SpoIIID is also subject to a developmentally regulated C- terminal truncation that alters its activity and stability. The resulting 9kDa form of SpoIIID will be analyzed to determine its C- terminus. The effects of truncating the spoIIID gene so that it encodes the 9 kDa protein will be studied. The protein that converts SpoIIID to the 9 kDa form will be identified, the corresponding gene cloned, and th effects of a null mutation determined. SigmaK RNA polymerase transcribe the gene encoding the third component of the switch, GerE, which exerts the opposite effect of SpoIIID on the transcription of several genes. Mutational analyses of the binding sites for SpoIID and GerE in promoter where they activate or repress transcription, and biochemical experiments, will be used to explore how these proteins exert their copious effects on mother-cell gene transcription.

这项研究的长期目标是了解基因 表达在相对的时间内由时间和空间控制 枯草芽孢杆菌孢子形成的简单发育过程。 这 过程涉及一个高度有序的基因表达程序和 形态学变化，并为 阐明基因调节的基本机制。 这些研究AR 具有广泛的意义，将对公众产生重要意义 健康，因为基因表达在时间和空间上受到调节 在细胞周期以及自适应和发育期间 反应，包括病原体与其宿主的相互作用。 拟议的研究集中于三个关键调节器，包括 开关管理从早期基因表达的过渡 发育中的孢子囊的母细胞室。开关是 响应于来自另一个隔室的信号 孢子囊，前孔。 此步骤涉及处理不活动 前体Pro-Sigmak，to Sigmak，sigmak，一个RNA聚合酶的亚基， 指导母细胞中的晚期基因转录。 SpoIVFB蛋白 可能是处理sigmak的蛋白酶，或 处理反应。 为了阐明处理机制， 将开发并使用用于SPOIVFB的抗体探针来确定其 细胞内位置，以及分离富集的分数 SPOIVFB，将用于重建Pro-Sigmak处理 体外。 绕过对孢子体中spoiVFB的突变将 被认为是确定SpoiVFB是否可能是Pro- Sigmak加工酶，如果没有，则识别蛋白酶。 这 Sigmak的出现在某种程度上导致Sigmae水平降低， 指导早期母细胞基因转录。 这停止了 SpoIIID的成绩单，它编码了该的第二个关键组件 转变。 SpoIIID蛋白激活或抑制机器人的转录 早期和晚母细胞基因。 SIGK中的突变（编码Sigmak） 将被构造并用于确定Sigmak是否必须是 转录活性至负调节Sigmae和Spoiiid 水平。 SpoiiID还受到发育监管的C- 末端截断会改变其活性和稳定性。 这 将分析结果的9KDA形式的spoiiid，以确定其c- 终点。 截断Spoiiid基因的影响，以使其编码 将研究9 kDa蛋白。 将spoiiid转换为的蛋白质 将确定9 kDa形式，相应的基因克隆，然后 确定无效突变的作用。 Sigmak RNA聚合酶转录 编码开关GERE的第三个组成部分的基因，该基因发挥作用 Spoiiid对几种基因转录的相反作用。 启动子中SpoIID和GERE的结合位点的突变分析 它们激活或抑制转录以及生化 实验将用于探索这些蛋白质如何施加 对母细胞基因转录的大量影响。