SWITCH GOVERNING BACILLUS MOTHER CELL GENE EXPRESSION

这项研究的长期目标是了解基因是如何 在相对的时间和空间上，表达受到控制 枯草芽孢杆菌产孢子的简易发育过程。这 这个过程涉及一个高度有序的基因表达程序和 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。这些研究包括 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是受调控的 在细胞周期中，以及在适应和发育期间 反应，包括在病原体与其宿主相互作用期间。 拟议的研究集中在三个关键监管机构上，这三个监管机构包括 控制基因表达从早期到晚期的转换 发育中的孢子囊的母细胞室。该开关是 响应来自另一个隔间的信号而启动 孢子囊，前孔。此步骤涉及处理非活动的 SigmaK的前体，Pro-SigmaK，RNA聚合酶的一个亚单位， 在母细胞中指导晚期基因转录。SpoIVFB蛋白 可能是处理Pro-sigmaK的蛋白酶，或者是 处理反应。为了阐明加工的机制，一种 将开发SpoIVFB抗体探针并用于检测其 细胞内定位，以及分离富集组分 SpoIVFB，它将用于重建Pro-sigmaK处理 体外培养。在产孢子过程中绕过SpoIVFB的突变将 以确定SpoIVFB是否可能是 SigmaK加工酶，如果不是，则鉴定该蛋白酶。这个 SigmaK的出现以某种方式导致SigmaE水平下降， 它指导着早期母细胞基因的转录。这会停顿下来 SpIIID的转录本，它编码了第二个关键成分 换个位置。SpoIIid蛋白激活或抑制BOT转录 早期和晚期的母细胞基因。SigK(编码sigmaK)的突变 将被构造并用于确定sigmaK是否必须 转录活性负调控SigmaE和SpoIIID 级别。SpoIIID还受发育调节的C- 改变其活动性和稳定性的末端截断。这个 得到的9 kDa形式的SpoIIID将被分析以确定其C- 终点站。截短SPIIID基因以使其编码的效果 将对9 kDa蛋白质进行研究。将SpoIIid转化为 将鉴定9 kDa形式，克隆相应的基因，并 确定了零突变的影响。SigmaK RNA聚合酶转录 编码开关的第三个组件Gere的基因，它施加 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和生化 实验，将被用来探索这些蛋白质是如何发挥其 对母细胞基因转录有丰富的影响。

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.