A- and T-tract-mediated intrinsic curvature in native DNA between the binding site of the upstream activator NtrC and the nifLA promoter of Klebsiella pneumoniae facilitates transcription.
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The nif promoters of Klebsiella pneumoniae must be activated by proteins bound to upstream sequences which are thought to interact with the sigma54-RNA polymerase holoenzyme by DNA looping. NifA is the activator for most of the promoters, and integration host factor (IHF) mediates the DNA looping. While NtrC is the activator for the nifLA promoter, no IHF appears to be involved. There are two A tracts and one T tract between the upstream enhancer and the nifLA promoter. This DNA segment exhibits anomalous electrophoretic mobility, suggesting intrinsic sequence-induced curvature in the DNA. On the one hand, mutation of the A tracts or T tract individually or together, or deletion of the A tracts and the T tract reduces the anomaly; on the other hand, creation of two additional A tracts enhances the anomaly. Intrinsic curvature in the DNA has been confirmed by circular permutation analysis after cloning the DNA fragment in the vector pBend 2 and also by electron microscopy. Computer simulation with the DNA base sequence is also suggestive of intrinsic curvature. A transcriptional fusion with the Escherichia coli lacZ gene of the DNA fragment containing the nifLA promoter and the wild-type or the mutated upstream sequences was constructed, and in vivo transcription in K. pneumoniae and E. coli was monitored. There was indeed very good correlation between the extent of intrinsic curvature of the DNA and transcription from the promoter, suggesting that DNA curvature due to the A tracts and the T tract was necessary for transcription in vivo from the nifLA promoter of K. pneumoniae. (+info)
Mutations affecting motifs of unknown function in the central domain of nitrogen regulatory protein C.
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The positive control function of the bacterial enhancer-binding protein NtrC resides in its central domain, which is highly conserved among activators of sigma54 holoenzyme. Previous studies of a small set of mutant forms specifically defective in transcriptional activation, called NtrC repressor [NtrC(Rep)] proteins, had enabled us to locate various functional determinants in the central domain. In this more comprehensive survey, the DNA encoding a major portion of the central domain was randomly mutagenized and mutated ntrC genes were introduced into the cell via multicopy expression plasmids. DNA sequencing of 95 isolates identified by a preliminary phenotypic screen revealed that the lesions in them caused 55 distinct single amino acid substitutions at 44 different positions. Assays of glnA transcription in vivo and in vitro yielded two conclusions. First, of the 41 mutant proteins that could be purified, 17 (1 known, 16 new) showed no detectable activity in either assay, thus qualifying them as true NtrC(Rep) proteins. These contained residue changes in six of the seven highly conserved regions in the central domain, including two never studied before. Second, some mutant proteins were inactive in vivo but were either marginally or fully active in vitro. Their surprising lack of activity in vivo may be accounted for by high levels of expression, which apparently decreased activation by these mutant proteins but not by wild-type NtrC (NtrCWT). Of particular interest were a subset of these proteins that exhibited greater transcriptional activation than NtrCWT at low concentrations. Their elevated activation capacities remain to be explained. (+info)
Purification of P(II) and P(II)-UMP and in vitro studies of regulation of glutamine synthetase in Rhodospirillum rubrum.
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The P(II) protein from Rhodospirillum rubrum was fused with a histidine tag, overexpressed in Escherichia coli, and purified by Ni(2+)-chelating chromatography. The uridylylated form of the P(II) protein could be generated in E. coli. The effects on the regulation of glutamine synthetase by P(II), P(II)-UMP, glutamine, and alpha-ketoglutarate were studied in extracts from R. rubrum grown under different conditions. P(II) and glutamine were shown to stimulate the ATP-dependent inactivation (adenylylation) of glutamine synthetase, which could be totally inhibited by alpha-ketoglutarate. Deadenylylation (activation) of glutamine synthetase required phosphate, but none of the effectors studied had any major effect, which is different from their role in the E. coli system. In addition, deadenylylation was found to be much slower than adenylylation under the conditions investigated. (+info)
Expression of P(II) and glutamine synthetase is regulated by P(II), the ntrBC products, and processing of the glnBA mRNA in Rhodospirillum rubrum.
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We have studied the transcription of the glnB and glnA genes in Rhodospirillum rubrum with firefly luciferase as a reporter enzyme. Under NH(4)(+) and N(2) conditions, glnBA was cotranscribed from a weak and a strong promoter. In nitrogen-fixing cultures, activity of the latter was highly enhanced by NtrC, but transcription from both promoters occurred under both conditions. There is no promoter controlling transcription of glnA alone, supporting our proposal that the glnA mRNA is produced by processing. (+info)
"Switch I" mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA.
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NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein of 469 residues that activates transcription by sigma(54)-holoenzyme. A region of its transcriptional activation (central) domain that is highly conserved among homologous activators of sigma(54)-holoenzyme-residues 206-220-is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydrolysis to a change in the conformation of the polymerase that allows it to form transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrC(A216V) and NtrC(G219K), have normal ATPase activity but fail in transcriptional activation. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrC(A216C) and NtrC(G219C), are capable of activating transcription when they are not bound to a DNA template (non-DNA-binding derivatives with an altered helix-turn-helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA remains a positive allosteric effector of ATP hydrolysis, as it is for wild-type NtrC but, surprisingly, appears to have become a negative allosteric effector for some aspect of interaction with sigma(54)-holoenzyme. The conserved region in which these amino acid substitutions occur (206-220) is equivalent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region of NtrC and that of p21(ras). (+info)
General nitrogen regulation of nitrate assimilation regulatory gene nasR expression in Klebsiella oxytoca M5al.
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Klebsiella oxytoca can assimilate nitrate and nitrite by using enzymes encoded by the nasFEDCBA operon. Expression of the nasF operon is controlled by general nitrogen regulation (Ntr) via the NtrC transcription activator and by pathway-specific nitrate and nitrite induction via the NasR transcription antiterminator. This paper reports our analysis of nasR gene expression. We constructed strains bearing single-copy Phi(nasR-lacZ) operon fusions within the chromosomal rhaBAD-rhaSR locus. The expression of DeltarhaBS::[Phi(nasR-lacZ)] operon fusions was induced about 10-fold during nitrogen-limited growth. Induction was reduced in both ntrC and rpoN null mutants, indicating that Ntr control of nasR gene expression requires the NtrC and sigma(N) (sigma(54)) proteins. Sequence inspection of the nasR control region reveals an apparent sigma(N)-dependent promoter but no apparent NtrC protein binding sites. Analysis of site-specific mutations coupled with primer extension analysis authenticated the sigma(N)-dependent nasR promoter. Fusion constructs with only about 70 nucleotides (nt) upstream of the transcription initiation site exhibited patterns of beta-galactosidase expression indistinguishable from Phi(nasR-lacZ) constructs with about 470 nt upstream. Expression was independent of the Nac protein, implying that NtrC is a direct activator of nasR transcription. Together, these results indicate that nasR gene expression does not require specific upstream NtrC-binding sequences, as previously noted for argT gene expression in Salmonella typhimurium (G. Schmitz, K. Nikaido, and G. F.-L. Ames, Mol. Gen. Genet. 215:107-117, 1988). (+info)
Protein PII regulates both inorganic carbon and nitrate uptake and is modified by a redox signal in synechocystis PCC 6803.
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In Synechocystis PCC 6803 as in other cyanobacteria, involvement of protein PII in the co-regulation of inorganic carbon and nitrogen metabolism was established based on post-translational modifications of the protein resulting from changes in the carbon/nitrogen regimes. Uptake of bicarbonate and nitrate in response to changes of the carbon and/or nitrogen regimes is altered in a PII-null mutant, indicating that both processes are under control of PII. Modulation of electron flow by addition of methyl viologen with or without duroquinol, or in a NAD(P)H dehydrogenase-deficient mutant, affects the phosphorylation level of PII. The redox state of the cells would thus act as a trigger for PII phosphorylation. (+info)
Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators.
(16/281)
Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains. (+info)