ANAEROBIC EXPRESSION OF FUMARATE REDUCTASE IN E. COLI

这项研究的目的是为了更清楚地了解 富马酸盐的诱导、合成和周转机制 还原酶复合物。 这种氧化还原酶复合物 是膜结合的，含有黄素蛋白和非血红素铁 中心. 富马酸盐的四种单独多肽的合成 还原酶复合物仅在厌氧生长条件下发生， 富马酸盐可用作末端电子受体。 合成 当氧或硝酸盐存在时，络合物的活性被抑制。 之甚少 已知这种诱导和/或抑制现象，无论是在 基因或分子水平。 我们计划研究转录调控的机制， 四个基因编码膜结合的单个多肽， 富马酸还原酶复合物。 导致延胡索酸还原酶表达改变的突变以及 将产生延胡索酸还原酶的结构基因突变 增加我们对监管现象的理解， 复杂的生物化学。 在体内表达和周转， 在诱导和非诱导条件下的富马酸还原酶复合物将 也可以使用基因融合和免疫学技术进行检查。 影响frd表达的富马酸盐、硝酸盐和FNR调节蛋白 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 通过阳性调节蛋白FNR进行表达。 编码FNR的fnr基因的转录调控将被研究。 检查以定位启动子和相关的调节控制区。 我们认为，延胡索酸还原酶是一个很好的模型系统， 基因的诱导和调节，其产物是在 厌氧生长条件 关于这些基因是如何 对富马酸盐、硝酸盐和氧的反应被诱导或抑制。 虽然 对E.大肠杆菌本身就很有趣， 这个系统也可以作为一个有用的模型来理解兼性 其他肠道细菌的代谢。

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.