The long term goal of the proposed research is to determine molecular details regarding the regulation of a fatty acid biosynthetic operon of Escherichia coli K-12. The operon to be characterized in detail is the orf1-rpmF-plsX-fabH-fabD-fabG-acpP operon which encodes protein L32 of the large ribosomal subunit (rpmF), 3-ketoacyl-acyl carrier protein synthase III (fabH), malonyl CoA-acyl carrier protein (ACP) transacylase (fabD), 3-ketoacyl-ACP reductase (fabG), ACP (acpP) and two proteins of unknown function (Orf1 and PlsX). Although other operons which encode ribosomal proteins contain genes for essential cellular processes including DNA replication, transcription and translation, this is the only known example of lipid biosynthetic genes which are cotranscribed with a ribosomal protein gene. The juxtaposition of these genes is likely to play a role in the coordinate regulation of the synthesis of ribosomes and the cell membranes. The locations of the multiple promoters in this operon will be determined. The effects of polar insertions in each of the genes on expression of downstream genes will be assessed. The transcripts originating from promoters or RNA processing will be identified by Northern blotting. The 5' and 3' ends of the transcripts will be mapped by S1 nuclease and primer extension analysis. The 5' ends generated by RNA processing will be distinguished from those generated by promoter activity by using an RNA capping assay. Gene dosage experiments will be performed to determine if expression of the operon is autoregulated by the Orf1, L32 or PlsX proteins. The regulatory role played by an attenuator located between rpmF and plsX will be determined by quantitation of transcripts encoded by regions upstream and downstream of the attenuator. Fusions between the rpmF, plsX and fabH genes and lacZ will be constructed and used to determine if the ribosomal protein gene rpmF and the fatty acid biosynthetic genes are coordinately regulated during steady state growth conditions and during metabolic perturbations of growth rate, such as those that occur during the stringent response. Finally, the biochemical function of the plsX gene product will be determined by using biochemical and genetic approaches. This protein has been implicated in the incorporation of fatty acids into the membrane phospholipids, or in fatty acid biosynthesis, or in the regulation of these processes.
