ASSEMBLY OF A MEMBRANE-BOUND MULTI-ENZYME COMPLEX

我们建议扩大对基因表达调控的研究。 NAR操纵子与细菌硝酸还原酶复合体的生物发生 大肠杆菌的一般目的如下：(A)界定 诱导特定基因表达的分子机制 厌氧条件和特定基因对该基因表达的调节 电子受体和(B)定义时间序列和分子 膜结合的功能性细胞的生物发生机制 很复杂。 硝酸还原酶是大肠杆菌中的一种膜结合酶 允许这种兼性厌氧菌利用硝酸盐作为电子进行生长 厌氧条件下的受体。这种酶是在 厌氧条件和一系列酶使E。 利用发酵或厌氧呼吸来获取能量 外源电子受体。Nar操纵子的表达，它 编码硝酸还原酶，在转录水平上受 一种多种厌氧表达酶的正调节因子， FNR，以及一个特定的积极监管因子NarL，它是 被硝酸盐的存在所激活的。我们建议研究分子 FNR厌氧活化及活化机理的研究 这两个反式作用系统对nar操纵子转录的影响。 膜结合硝酸还原酶由三个亚基组成(α， β)、钼-翼状体辅因子、非血红素离子和细胞色素 B组分。这种复合体在细胞膜中被组织起来，从而 硝酸盐还原为亚硝酸盐与发酵氧化耦合 途径中间体或产物驱动质子的形成 渐变。α和β亚基形成一种复合体，它可以减少 只有人工给电子体的硝酸盐，而伽马亚基是 明显锚定络合物的疏水性细胞色素b组分 以生理功能的形式连接到膜上。我们建议 利用克隆的nar操纵子和不能产生的突变体 确定时间事件和分子的特定亚基 亚基合成、辅因子加成和组装的机制 细胞膜中的功能复合体。

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.