ANAEROBIC EXPRESSION OF FUMARATE REDUCTASE IN E. COLI

这项研究建议的目的是为了更清楚地了解 富马酸酯的诱导、合成和转化机理 大肠杆菌中的还原酶复合体。这种氧化还原酶复合体 是膜结合的，同时含有黄素蛋白和非血红素铁 中锋。富马酸四个单体多肽的合成 只有在厌氧生长条件下才会出现还原酶复合体 富马酸盐可用作终端电子受体。合成法 当有氧气或硝酸盐存在时，复合体的作用被抑制。小才是 已知这种诱导和/或压抑现象 在基因或分子水平上。 我们计划研究转录调控的机制。 编码膜结合的单个多肽的四个基因 利用重组DNA技术合成富马酸还原酶复合体。 导致富马酸还原酶表达改变的突变以及 富马酸还原酶的结构基因将产生突变 增加我们对监管现象和 复合体的生物化学。该蛋白在体内的表达和周转 富马酸还原酶复合体在诱导和非诱导条件下将 也可以使用基因融合和免疫学技术进行检测。 富马酸、硝酸盐和FNR调节蛋白对Frd表达的影响 将使用遗传和分子方法进行研究。实验 旨在了解富马酸还原酶的激活 将进行正调控蛋白FNR的表达。 编码FNR的FNR基因的转录调控将是 检查以定位启动子和相关的调控区域。 我们认为富马酸还原酶是一个很好的研究模型系统。 对其产物需要的基因的诱导和调控 厌氧生长条件。关于这些基因是如何形成的，我们知之甚少。 对富马酸、硝酸盐和氧气的反应被诱导或抑制。虽然 富马酸还原酶在大肠杆菌中的研究本身就很有趣， 这个系统也可以作为理解兼职的一个有用的模型。 其他肠道细菌的新陈代谢。

The aim of this research proposal is to gain a clearer understanding of the mechanisms for the induction, synthesis and turnover of the fumarate reductase complex in Escherichia coli. This oxido-reductase enzyme complex is membrane bound and contains both flavoprotein and non-heme iron centers. Synthesis of the four individual polypeptides of the fumarate reductase complex occurs only under anaerobic growth conditions when fumarate is available for use as a terminal electron acceptor. Synthesis of the complex is repressed when oxygen or nitrate is present. Little is known about this induction and/or repression phenomenon at either the genetic or the molecular level. We plan to examine the mechanisms for the transcriptional regulation of the four genes encoding the individual polypeptides of the membrane bound fumarate reductase complex using recombinant DNA techniques. Mutations resulting in altered fumarate reductase expression as well as mutations in the structural genes for fumarate reductase will be generated to increase our understanding of both the regulatory phenomenon and biochemistry of the complex. The in vivo expression and turnover of the fumarate reductase complex under inducing and non inducing conditions will also be examined using gene fusion and immunological techniques. The fumarate, nitrate and FNR regulator proteins affecting frd expression will be studied using genetic and molecular approaches. Experiments directed towards understanding the activation of fumarate reductase expression by the positive regulatory protein, FNR, will be undertaken. The transcriptional regulation of the fnr gene encoding FNR will be examined to locate the promoter and associated regulatory control regions. We believe that fumarate reductase is an excellent model system to examine the induction and regulation of genes whose products are required under anaerobic growth conditions. Little is known about how these genes are induced or repressed in response to fumarate, nitrate and oxygen. Although the study of fumarate reductase in E. coli is interesting in its own right, this system may also serve as a useful model for understanding facultative metabolism in other enteric bacteria.