| Predictions of gene regulation from DNA sequences are generally based on the poorly-tested hypothesis that a conserved regulator, conserved target gene, and putative binding sites for the regulator upstream of the target gene, imply a conserved pattern of regulation. Inherent to this framework is the assumption that well-conserved regulators are intrinsically and extrinsically equivalent, a presumption that has not been fully tested.; The equivalency of the leucine-responsive regulatory protein (Lrp) was assessed for three well-conserved orthologs from the gamma proteobacteria Escherichia coli, Proteus mirabilis and Vibrio cholerae. Using the well-defined E. coli system as a model, it was determined that the Lrp orthologs maintained their ability to properly regulate heterologous target genes in the presence and absence of a coregulator. In addition, the orthologous regulators functioned well as part of the complex response associated with hyperflagellated swarmer cell differentiation in the pathogen P. mirabilis. However, there were some differences in the effects of Lrp orthologs on the activity of various target promoters, suggesting differences in affinity. The regulatory control of the lrp gene itself, in addition to intrinsic differences between orthologs, was found to be vital to its regulatory scope. This was indicated by the variation in Proteus swarming phenotypes, and by the reduced number of target genes complemented by lrp under the control of Plac compared to the native promoter.; Experimental methods that identify the transcription factors associated with a particular promoter offer essential details about combinatorial regulatory control. An important limitation on whole-genome analysis of regulation is lack of a relatively high-throughput method for identifying regulatory proteins associated with a given promoter, without knowing in advance what any of those proteins are. In general, current methods either lack the ability to identify unknown regulators or lack the context of true in vivo conditions. A plasmid-based in vivo method for unknown regulator identification was proposed, and developed on a preliminary basis. The method allows enrichment of regulators associated with a given promoter, and may be useful in both prokaryotic and eukaryotic systems. |
