ASSEMBLY OF A MEMBRANE-BOUND MULTI-ENZYME COMPLEX

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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.