Binding of NorR to three DNA sites is essential for promoter activation of the flavorubredoxin gene, the nitric oxide reductase of Escherichia coli

NorR is a nitric oxide sensor that in Escherichia coli regulates the gene encoding for flavorubredoxin, an enzyme involved in nitrosative detoxification. The present work shows that although purified NorR can bind independently to each of three binding sites in the flavorubredoxin gene promoter, the presence of all sites is required for in vivo nitric oxide-dependent induction of the flavorubredoxin gene. Furthermore, trimerization of NorR upon binding to the three sites was observed by protein cross-linking experiments. These results reveal the importance of the multiple DNA binding sites present on NorR-dependent promoters and suggest that the functional form of NorR is a trimer.     

Characterization of the MetR binding sites for the glyA gene of Escherichia coli.

Sequence analysis of the glyA control region of Escherichia coli identified two regions with homology to the consensus binding sequence for MetR, a lysR family regulatory protein. Gel shift assays and DNase I protection assays verified that both sites bind MetR. Homocysteine, a coregulator for MetR, increased MetR binding to the glyA control region. The MetR binding sites were cloned into the pBend2 vector. Although the DNA did not show any significant intrinsic bend, MetR binding resulted in a bending angle of about 33 degrees. MetR-induced bending was independent of homocysteine. To verify that the MetR binding sites play a functional role in glyA expression, site-directed mutagenesis was used to alter the two binding sites in a lambda glyA-lacZ gene fusion phage. Changing the binding sites toward the consensus MetR binding sequence caused an increase in glyA-lacZ expression. Changing either binding site away from the consensus sequence caused a decrease in expression, suggesting that both sites are required for normal glyA regulation.     

Cyclic AMP receptor proteins interacts with lactose operator DNA

The wildtype lac promoter-operator region of E. coli was reinvestigated for cyclic AMP receptor protein (CRP) binding sites using the 'footprinting' method. Two distinct interaction sites were found, one in the -60 base pair region, which has previously been described, and a second one covering the lac operator. These two sites are similar in extent, orientation and the characteristic pattern of CRP-induced DNaseI enhanced cutting sites.     

The binding of cyclic AMP receptor protein to two lactose promoter sites is not cooperative in vitro.

The lactose promoter-operator region of Escherichia coli contains two binding sites for cyclic AMP receptor protein (CAP), two for the lactose repressor, and two for RNA polymerase. The high density of binding sites makes cooperative interactions between these proteins likely. In this study, we used the gel electrophoresis mobility shift assay and binding partition analysis techniques to determine whether the secondary CAP site influences the binding of CAP to the principal CAP site in the lactose promoter when both are present on a linear DNA molecule. Such an effect could occur through the formation of a bridged DNA-CAP-DNA structure, through the interaction of CAP molecules bound to each of the sites, or through allosteric effects caused by CAP-mediated DNA bending. We found, however, that the interaction of CAP with these sites was not cooperative, indicating that CAP sites 1 and 2 bind CAP in an independent manner.     

Interaction of integration host factor from Escherichia coli with the integration region of the Haemophilus influenzae bacteriophage HP1.

The specific DNA-binding protein integration host factor (IHF) of Escherichia coli stimulates the site-specific recombination reaction between the attP site of bacteriophage HP1 and the attB site of its host, Haemophilus influenzae, in vitro and also appears to regulate the expression of HP1 integrase. IHF interacts specifically with DNA segments containing the att sites and the integrase regulatory region, as judged by IHF-dependent retardation of relevant DNA fragments during gel electrophoresis. The locations of the protein-binding sites were identified by DNase I protection experiments. Three sites in the HP1 attP region bound IHF, two binding sites were present in the vicinity of the attB region, and one region containing three partially overlapping sites was present in the HP1 integrase regulatory segment. The binding sites defined in these experiments all contained sequences which matched the consensus IHF binding sequences first identified in the lambda attP region. An activity which stimulated the HP1 site-specific integration reaction was found in extracts of H. influenzae, suggesting that an IHF-like protein is present in this organism.     

Identification of cyclic AMP-CRP binding sites in the intercistronic regulatory uxaCA-exuT region of Escherichia coli

Abstract In Escherichia coli K-12 the two adjacent operons exuT and uxaCA are divergently transcribed and each possesses its own control region. We show that in vivo the expression of these two operons seems to be sensitive to catabolite repression and to be cAMP-dependent. The nucleotide sequence of 318 nucleotides, including the entire control region of uxaCA and the exuT operator, was determined and two cAMP-CRP binding sites were identified.     

Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal (Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.