IN-VITRO INTERACTION OF NITRATE-RESPONSIVE REGULATORY PROTEIN NARL WITH DNA TARGET SEQUENCES IN THE FDNG, NARG, NARK AND FRDA OPERON CONTROL REGIONS OF ESCHERICHIA-COLI K-12

The narL gene product is a nitrate-responsive activator and repressor of anaerobic respiratory gene expression. Mutational studies and seque nce comparisons have suggested that NarL protein binding sites contain heptameric sequences related to the consensus, TACYNMT (where Y=C or T, M=A or C, and N = any nucleotide). There are four NarL heptamers in the -105 region of the fdnGHI (formate dehydrogenase-N) operon, and m utational analysis supports the role of these heptamers in nitrate ind uction. To examine NarL-DNA interactions, we purified the NarL protein as a maltose binding protein (MBP) fusion protein (MBP-NarL). A const itutive mutant form with a single substitution (V88A) in the amino-ter minal (response regulator) region was used. The MBP-NarL(V88A) protein protected all four heptamers in the fdnG operon control region from D Nase I cleavage. Identical footprints were observed with NarL(V88A) pr otein that had been proteolytically cleaved free from the MBP domain. Binding of MBP-NarL(V88A) protein to the four heptamers in the -105 re gion of the fdnG operon appeared to be cooperative, and occupancy of t he central heptamers was necessary for occupancy of the flanking hepta mers. In addition to the V88A substitution, a low molecular weight pho sphodonor, such as acetyl phosphate, was required for observable footp rints. This indicates that phosphorylation of the NarL protein enhance s its affinity for its multiple DNA targets in the fdnG operon, perhap s by increasing protein-protein interactions rather than protein-DNA i nteractions. We also performed footprinting studies at the narGHJI (ni trate reductase), narK (nitrite efflux), and frdABCD (fumarate reducta se) operon control regions. Extensive areas of each control region wer e protected from DNase I attack by phosphorylated MBP-NarL(V88A) prote in. The narG operon control region was protected from positions -50 to -110, and, at higher protein concentrations, also around position -20 0. Mutational analysis indicates that the NarL heptamer centered at po sition -89, in addition to the previously-identified -200 region, is i nvolved in nitrate induction. Comparisons of the four operon control r egions studied indicate that the NarL heptamers are arranged with dive rse orientations and spacing.