Interactions of the leucine-responsive regulatory protein with the promoter region of the gltBDF operon of Escherichia coli specifying glutamate synthase.

Abstract

More than 40 genes and operons in <italic>Escherichia coli</italic> have been shown to be regulated by the global regulator Lrp (leucine-responsive regulatory protein), acting in conjunction with its co-regulator leucine. The level of sensitivity of operons to leucine varies greatly and the basis for this variation is only partially understood. One operon controlled by Lrp that is relatively insensitive to leucine is <italic>gltBDF</italic>, which includes genes specifying the large (GltB) and small (GltD) subunits of glutamate synthase. The aim of this work has been to better understand the roles played by Lrp and leucine in the transcriptional activation of <italic>gltBDF</italic> and to apply that understanding to create a model consistent with behavior of leucine and Lrp at other operons. To this end, a number of complementary studies were carried out: DNase I protection assays identified and mapped the location of three Lrp binding sites upstream of <italic>gItBDF</italic>; <super> 32</super>p-DNA/<super>35</super>S-Lrp double-label gel shift assays determined that three Lrp dimers bound to <italic>gltBDF</italic>; and atomic force microscopy revealed the presence of large nucleoprotein structures involving the entire binding region. In addition, the relative importance of the three sites in transcriptional activation was studied by means of a series of site-directed mutations which revealed (1) that all three sites and the proper phasing of these sites, with one another and with the start of transcription, are required for maximal expression and (2) that leucine, while decreasing the affinity of Lrp for DNA, increases the efficacy of Lrp that is bound. The effects of these mutations were determined by DNase I footprinting, by quantitative gel shift assays, and by measuring b -galactosidase activity levels of cells with chromosomal <italic>gltB </italic>::<italic>lacZ</italic> operon fusions. Ultimately, this work has led to two major findings. First, it has yielded a clear definition and characterization of the <italic>gltBDF</italic> promoter. Second, it suggests that the range in leucine sensitivity shown by various operons results not only from the cellular concentration of Lrp and the affinity Lrp has for a given promoter, but also from the competing effects that leucine has on Lrp binding affinity for DNA and on the ability of Lip to facilitate transcription once bound.