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Query: EC:1.7.1.2 (nitrate reductase)
 3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nitrate reductase operon (narGHJI) of Escherichia coli encodes an anaerobic respiratory enzyme. Previous work has identified two cis-acting sites in the nar operon control region: a proximal site required for anaerobic induction mediated by the activator Fnr and a remote upstream site required for nitrate induction mediated by the activator NarL [Li, S. & DeMoss, J. A. (1988) J. Biol. Chem. 263, 13700-13705]. Our search for nar regulatory mutants yielded one strain with a mutation in himD, the structural gene for one of the subunits of integration host factor (IHF). Strains carrying null alleles of the IHF structural genes, himD and himA, had severe defects in nitrate induction of the nar operon but were normal for nitrate induction of the coordinately regulated fdn operon. Anaerobic expression of both operons was normal in him mutants. Gel-mobility-shift and DNase I protection experiments revealed a single IHF binding site in the nar operon control region, located midway between the upstream activation site and the promoter. We conclude that an IHF-mediated DNA bend is essential for efficient nitrate induction of the sigma 70-dependent nar operon promoter. This requirement of IHF for transcriptional activation had been noted for several sigma 54-dependent promoters.
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PMID:In vivo requirement of integration host factor for nar (nitrate reductase) operon expression in Escherichia coli K-12. 152 82

The narL gene product is a nitrate-responsive activator and repressor of anaerobic respiratory gene expression. Mutational studies and sequence 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 mutational analysis supports the role of these heptamers in nitrate induction. To examine NarL-DNA interactions, we purified the NarL protein as a maltose binding protein (MBP) fusion protein (MBP-NarL). A constitutive mutant form with a single substitution (V88A) in the amino-terminal (response regulator) region was used. The MBP-NarL (V88A) protein protected all four heptamers in the fdnG operon control region from DNase I cleavage. Identical footprints were observed with NarL (V88A) protein that had been proteolytically cleaved free from the MBP domain. Binding of MBP-NarL (V88A) protein to the four heptamers in the -105 region of the fdnG operon appeared to be cooperative, and occupancy of the central heptamers was necessary for occupancy of the flanking heptamers. In addition to the V88A substitution, a low molecular weight phosphodonor, such as acetyl phosphate, was required for observable footprints. This indicates that phosphorylation of the NarL protein enhances its affinity for its multiple DNA targets in the fdnG operon, perhaps by increasing protein-protein interactions rather than protein-DNA interactions. We also performed footprinting studies at the narGHJI (nitrate reductase), narK (nitrite efflux), and frdABCD (fumarate reductase) operon control regions. Extensive areas of each control region were protected from DNase I attack by phosphorylated MBP-NarL (V88A) protein. The narG operon control region was protected from positions -50 to -110, and, at higher protein concentrations, also around position -200. Mutational analysis indicates that the NarL heptamer centered at position -89, in addition to the previously-identified -200 region, is involved in nitrate induction. Comparisons of the four operon control regions studied indicate that the NarL heptamers are arranged with diverse orientations and spacing.
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PMID: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. 805 56

The putative overlapping consensus sequences (-129 to -105) for binding of fumarate nitrate reductase regulator- and integration host factor (IHF)-like proteins to puc operon upstream DNA of Rhodobacter sphaeroides was protected from DNase I digestion by purified Escherichia coli IHF. The binding of E. coli IHF to the purported IHF-binding site in the puc upstream DNA is highly sequence-specific. The recorded binding affinity was significantly lower than that of E. coli IHF to the lambda attP site. Employing site-directed changes in the DNA sequence within the -129 to -105 region, a loss in IHF binding, as monitored through gel retardation analysis, was correlated with alterations in puc operon expression monitored through the use of puc::lacZ transcriptional fusions. These results suggest that the IHF-binding site is involved in repression of puc operon transcription by oxygen as well as modulation of puc operon transcription levels by incident light intensity. Mutations specific to the upstream half of the putative fumarate nitrate reductase regulator-binding site of the puc upstream DNA did not show any physiological effects under the experimental conditions employed. Taken together, these studies reveal that the DNA sequence between -129 to -105 may involve facilitation of the interaction between upstream and downstream cis-acting regulatory sequences involved in puc operon expression.
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PMID:Transcriptional regulation of puc operon expression in Rhodobacter sphaeroides. Involvement of an integration host factor-binding sequence. 822 1

Integration host factor protein (IHF) was shown to be required for Fnr- and NarL-dependent activation of the nitrate reductase (narGHJI) operon of Escherichia coli in response to nitrate availability and anaerobiosis. Using a narG-lacZ reporter fusion to evaluate narGHJI expression in vivo both the nitrate and anaerobic dependent controls were severely impaired in a himA mutant compared with the wild type strain. IHF was also required for Fnr-independent anaerobic control of narGHJI expression. In vitro, purified IHF protein was shown to bind to a narG promoter fragment with an apparent dissociation value of 5 nM by use of a gel shift assay. DNase I footprinting studies revealed that IHF protects a 37-base pair region centered 125 base pairs 5' of the narG transcription site. These studies suggest that the IHF protein performs a DNA bending function at the narG promoter to allow nitrate-dependent activation by the NarL regulatory protein, and second, it enhances the Fnr-dependent expression from the narG promoter under anaerobic cell growth conditions. A model whereby three transcriptional activators, NarL, IHF, and Fnr, induce expression of a sigma 70-dependent promoter for the narGHJI operon is discussed.
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PMID:Activation of the Escherichia coli nitrate reductase (narGHJI) operon by NarL and Fnr requires integration host factor. 841 52

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.
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PMID:Fnr, NarP, and NarL regulation of Escherichia coli K-12 napF (periplasmic nitrate reductase) operon transcription in vitro. 969 69

Escherichia coli growing under anaerobic conditions produces several molybdoenzymes, such as formate hydrogenlyase (formate to H2 and CO2; hyc and fdhF genes) and nitrate reductase (narGHJI genes). Synthesis of these molybdoenzymes, even in the presence of the cognate transcriptional activators and effectors, requires molybdate in the medium. Besides the need for molybdopterin cofactor synthesis, molybdate is also required for transcription of the genes encoding these molybdoenzymes. In E. coli, ModE was previously identified as a repressor controlling transcription of the operon encoding molybdate transport components (modABCD). In this work, the ModE protein was also found to be a required component in the activation of hyc-lacZ to an optimum level, but only in the presence of molybdate. Mutant ModE proteins which are molybdate-independent for repression of modA-lacZ also restored hyc-lacZ expression to the wild-type level even in the absence of molybdate. Nitrate-dependent enhancement of transcription of narX-lacZ was completely abolished in a modE mutant. Nitrate-response by narG-lacZ and narK-lacZ was reduced by about 50% in a modE mutant. DNase I footprinting experiments revealed that the ModE protein binds the hyc promoter DNA in the presence of molybdate. ModE-molybdate also protected DNA in the intergenic region between narXL and narK from DNase I hydrolysis. DNA sequences (5' TAYAT 3' and 5' GTTA 3') found in ModE-molybdate-protected modABCD operator DNA were also found in the ModE-molybdate-protected region of hyc promoter DNA (5' GTTA-7 bp-CATAT 3') and narX-narK intergenic region (5' GTTA-7 bp-TACAT 3'). Based on these results, a working model is proposed in which ModE-molybdate serves as a secondary transcriptional activator of both the hyc and narXL operons which are activated primarily by the transcriptional activators, FhlA and NarL, respectively.
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PMID:Transcriptional regulation of molybdoenzyme synthesis in Escherichia coli in response to molybdenum: ModE-molybdate, a repressor of the modABCD (molybdate transport) operon is a secondary transcriptional activator for the hyc and nar operons. 1020 9

Regulation of transcription of the Escherichia coli moe operon, which codes for proteins connecting molybdate metabolism, molybdopterin synthesis, and apomolybdoenzyme synthesis, was investigated. Expression of the moe operon was independent of genes coding for molybdate transport and Mo-cofactor biosynthesis. Expression of moeA-lacZ increased during anaerobic growth (2.5-fold over the aerobic value) and in the presence of nitrate and trimethylamine N-oxide (3.5- and 1.5-fold, respectively). The nitrate-dependent increase in moe expression required the NarL protein, while the anaerobiosis-dependent increase in moeA-lacZ expression required Arc proteins. ArcA-phosphate and not ArcA bound to the DNA upstream of moe, shifted the electrophoretic mobility of moe promoter DNA, and protected the DNA from DNase I hydrolysis. Nitrate-independent transcription of moeA-lacZ was repressed by the FNR protein, which also protected moe operator DNA from DNase I hydrolysis. These results show that ArcA-phosphate and FNR have opposite effects on the transcriptional regulation of the moe operon, and the combined action of the two redox regulators modulate the level of Mo-cofactor in the cell. Apparently, the control of synthesis of Mo-cofactor and the apomolybdoenzymes nitrate reductase and trimethylamine N-oxide reductase are coupled at the level of the moe operon.
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PMID:Transcriptional regulation of the moe (molybdate metabolism) operon of Escherichia coli. 1135 10