ASSEMBLY OF A MEMBRANE-BOUND MULTI-ENZYME COMPLEX

我们建议扩大我们的研究，对表达的调控， nar操纵子和硝酸还原酶复合物的生物发生 大肠杆菌的一般目的如下：（a）定义 诱导特异性基因表达的分子机制 厌氧条件和特异性的 电子受体和（B）定义时间序列和分子 参与功能性膜结合细胞生物发生的机制 复杂. 硝酸还原酶是大肠杆菌中的膜结合酶， 允许这种兼性厌氧菌利用硝酸盐作为电子生长 在厌氧条件下的受体。这种酶是在 厌氧条件沿着酶的网络，其允许E. 大肠杆菌通过发酵或厌氧呼吸获得能量， 外源电子受体 nar操纵子的表达， 编码硝酸还原酶，在转录水平上受 许多厌氧表达酶的正调节剂， Fnr，以及一个特定的正调控因子，NarL，这是 被硝酸盐激活。 我们建议研究 参与Fnr厌氧活化的机制和Fnr的活化 这两个反式作用系统对nar操纵子的转录。 膜结合硝酸还原酶由三个亚基（α， β，γ），一种腺嘌呤-嘌呤辅因子，非血红素离子和一种细胞色素 B组分。 这种复合物在细胞膜中被组织起来， 将硝酸盐还原为亚硝酸盐，再加上发酵的氧化作用 路径中间体或产物驱动质子的形成 梯度离心α和β亚基形成复合物， 硝酸盐只与人工电子供体，而γ亚基是一个 疏水性细胞色素B组分，其显然锚定复合物 以生理功能的形式连接到膜上。 我们建议 利用克隆的nar操纵子沿着不能产生 特异性亚基来确定时间事件和分子 参与亚基合成、辅因子添加和组装的机制 细胞膜上的功能复合体。

We propose to extend our studies on the regulation of the expression of the nar operon and the biogenesis of the nitrate reductase complex in Escherichia coli with the following general aims: (a) To define the molecular mechanisms involved in induction of specific gene expression by anaerobic conditions and the modulation of that expression by specific electron acceptors and (b) to define the temporal sequence and molecular mechanisms involved int he biogenesis of the functional, membrane-bound complex. Nitrate reductase is a membrane-bound enzyme in Escherichia coli which permits this facultative anaerobe to grow using nitrate as an electron acceptor under anaerobic conditions. This enzyme is induced under anaerobic conditions along with a network of enzymes which permits E. coli to derive energy by fermentation or by anaerobic respiration using exogenous electron acceptors. The expression of the nar operon, which encodes nitrate reductase, is regulated at the level of transcription by a positive regulator of a number of anaerobically expressed enzymes, Fnr, as well as a specific positive regulatory factor, NarL, which is activated by the presence of nitrate. We propose to study the molecular mechanisms involved in the anaerobic activation of Fnr and the activation of transcription of the nar operon by these two trans-acting systems. Membrane-bound nitrate reductase is composed of three subunits (alpha, beta, gamma), a molybdenum-pterium cofactor, nonheme ion and a cytochrome b component. this complex is organized in the cell membrane so that reduction of nitrate to nitrite coupled to oxidation of fermentation pathway intermediates or products drives the formation of a proton gradient. The alpha and beta subunits form a complex which can reduce nitrate only with artificial electron donors while the gamma subunit is a hydrophobic cytochrome b component which apparently anchors the complex to the membrane in a physiologically functional form. We propose to utilize the cloned nar operon along with mutants unable to produce specific subunits to determine the temporal events and molecular mechanisms involved in subunit synthesis, cofactor addition and assembly of the functional complex in the cell membrane.