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: 2022361
• 负责人: LEE R KROOS
• 金额: $ 21.59万
• 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 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022361
• 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是如何 表达在时间上和空间上在相对的时间和空间上被控制。 枯草芽孢杆菌芽孢形成的简单发育过程。 这 这一过程涉及基因表达的高度有序程序， 形态变化，并提供了一个有吸引力的实验系统， 阐明基因调控的基本机制。 这些研究是 具有广泛的意义，并将对公众产生重要影响 健康，因为基因表达在时间和空间上受到调节， 在细胞周期中，以及在适应和发育过程中， 反应，包括在病原体与其宿主的相互作用期间。 拟议的研究集中在三个关键监管机构，包括一个 控制从早期到晚期基因表达转变的开关， 孢子囊发育中的母细胞室。开关 响应于来自所述第一隔室的另一隔室的信号而启动所述第二隔室。 孢子囊，前孢子。 此步骤涉及处理非活动的 sigmaK的前体，pro-sigmaK，sigmaK是RNA聚合酶的一个亚基， 指导母细胞中的晚期基因转录。 SpoIVFB蛋白 可以是处理pro-sigmaK的蛋白酶，或其调节剂。 加工反应 为了阐明加工机制， SpoIVFB的抗体探针将被开发并用于确定其 细胞内定位，以及分离富集 SpoIVFB，将用于重建pro-sigmaK处理， 体外 在孢子形成中绕过SpoIVFB需要的突变将 以确定SpoIVFB是否可能是亲- sigmaK加工酶，如果没有，则鉴定蛋白酶。 的 sigmaK的出现以某种方式导致sigmaE水平的降低， 其指导早期母细胞基因转录。 这个暂停 spoIIID的转录本，其编码第二个关键组分， 开关. SpoIIID蛋白激活或抑制bot的转录， 早期和晚期母细胞基因。 sigK突变（编码sigmaK） 将被构造并用于确定sigmaK是否必须 转录活性以负调节sigmaE和SpoIIID 程度. SpoIIID也受到发育调节的C- 末端截短改变其活性和稳定性。 的 将分析得到的9 kDa形式的SpoIIID以确定其C- 终点站 截短spoIIID基因使其编码 将研究9 kDa蛋白。 将SpoIIID转化为 将鉴定9 kDa形式，克隆相应的基因， 确定无效突变的影响。 SigmaK RNA聚合酶转录 编码开关第三种成分GerE的基因， SpoIIID对几个基因转录的相反作用。 启动子中SpoIID和GerE结合位点的突变分析 在那里它们激活或抑制转录， 实验，将用于探索这些蛋白质如何发挥其 对母细胞基因转录的影响。