The recent explosion in genomic sequence information has now made it possible to know all, or at least a large proportion, of the proteins produced by a given organism (i.e., its "proteome"). However, these organisms do not live in static environments, and the differential expression of specific proteins under different conditions can result in profound physiological differences between two genetically-identical cells in distinct environments. The next major challenge in meaningfully understanding genomic information is to understand not only gene- and operon-specific regulation, but also how regulation is integrated so as to give a coordinated response to environmental changes. To study global regulation, this project focuses on a model regulatory protein in a model organism; it studies global regulation of metabolism in Escherichia coli, focusing on regulation involving the leucine-responsive regulatory protein (Lrp). E. coil is an ideal model organism, since so much is known about it and the sequence of its genome has been determined. Lrp is a good model regulator for these studies as it controls so many genes and operons and can generate diverse patterns of regulation.
Genes that are regulated by Lrp will be identified by genetic methods and by a method that combines two-dimensional (2-D) gel electrophoresis and mass spectrometry. Refinement of the methodology to identify polypeptides in 2-D gels using mass spectrometry is expected to contribute significantly to analysis of proteomes. In another use of 2D gel electrophoresis, sequential analyses of protein expression following shifts in growth conditions will be used to monitor changes in the patterns of protein expression in isogenic strains containing or lacking Lrp. In this way, the contribution of Lrp to regulation of protein expression following exposure of cells to high osmolarity environments or to entry into stationary phase can be assessed, and additional proteins whose expression is regulated by Lrp may be identified.
In addition to these studies of the global roles of Lrp, studies of its molecular mechanism of transcriptional activation will also be continued. These studies have focused on regulation by Lrp of the gltBDF operon, which specifies an enzyme central to ammonia assimilation -- glutamate synthase. Employing hybrids between the well-characterized lacUV5 promoter region from the lac operon and the gltBDF promoter region, the elements necessary for Lrp-dependent transcription of a gene will be defined in vivo. Once the in vivo studies are complete, the results will be used to develop an in vitro transcription system for gltBDF. The nucleoprotein complex formed when Lrp binds the gltBDF promoter region has been visualized using scanning force microscopy, and these studies will continue. In vitro transcription and scanning force microscopy will synergistically test models for transcriptional regulation by Lrp that have been developed during the previous periods of NSF support.
These studies should illuminate the means by which a simple dimeric helix-turn-helix DNA-binding protein can alter the expression of so many genes and operons in such a wide variety of ways. Lrp can either activate or repress transcription of target genes, and its co-regulator leucine can alter both its affinity for target promoters and its efficacy as an activator or repressor once bound. These studies should, at a specific level, assist in understanding the role of Lrp and the genes it regulates in coordinating complex physiological responses of E. coli cells to changes in their environment. At a general level these studies should further understanding of how genomes actually function.
