Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During nitrogen-limited growth, transcription of glnA, which codes for glutamine synthetase, requires sigma 54-RNA polymerase and the phosphorylated from the nitrogen regulator I (NRI; also called NtrC). In cells in which the lac promoter controlled expression of the gene coding for NRI, increasing the intracellular concentration of NRI lowered the level of glutamine synthetase. The reduction in glutamine synthetase does not appear to result from the NRI-dependent sequestering of any protein that affects transcription of glnA. Our results also suggest that the negative effect of a high concentration of NRI on glnA expression is a major determinant of the level of glutamine synthetase activity in nitrogen-limited cells of a wild-type strain. We propose that the inhibition results from an impairment of the interaction between NRI-phosphate and RNA polymerase that stimulates glnA transcription. We discuss a model that can account for this reduction in glutamine synthetase.
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PMID:Role of nitrogen regulator I (NtrC), the transcriptional activator of glnA in enteric bacteria, in reducing expression of glnA during nitrogen-limited growth. 134 10

In vivo dimethyl sulfate footprinting of the Bacillus subtilis glnRA regulatory region under repressing and derepressing conditions demonstrated that the GlnR protein, encoded by glnR, interacts with two sites situated within and adjacent to the glnRA promoter. One site, glnRAo1, between positions -40 and -60 relative to the start point of transcription, is a 21-bp symmetrical element that has been identified as essential for glnRA regulation (H. J. Schreier, C. A. Rostkowski, J. F. Nomellini, and K. D. Hirschi, J. Mol. Biol. 220:241-253, 1991). The second site, glnRAo2, is a quasisymmetrical element having partial homology to glnRAo1 and is located within the promoter between positions -17 and -37. The symmetry and extent of modifications observed for each site during repression and derepression indicated that GlnR interacts with the glnRA regulatory region by binding to both sites in approximately the same manner. Experiments using potassium permanganate to probe open complex formation by RNA polymerase demonstrated that transcriptional initiation is inhibited by GlnR. Furthermore, distortion of the DNA helix within glnRAo2 occurred upon GlnR binding. While glutamine synthetase, encoded by glnA, has been implicated in controlling glnRA expression, analyses with dimethyl sulfate and potassium permanganate ruled out a role for glutamine synthetase in directly influencing transcription by binding to operator and promoter regions. Our results suggested that inhibition of transcription from the glnRA promoter involves GlnR occupancy at both glnRAo1 and glnRAo2. In addition, modification of bases within the glnRAo2 operator indicated that control of glnRA expression under nitrogen-limiting (derepressing) conditions included the involvement of a factor(s) other than GlnR.
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PMID:Interaction of the Bacillus subtilis glnRA repressor with operator and promoter sequences in vivo. 134 63

The gene encoding glutamine synthetase (GS), glnA, was cloned from Azotobacter vinelandii on a 6-kb EcoRI fragment that also carries the ntrBC genes. The DNA sequence of 1,952 bp including the GS-coding region was determined. An open reading frame of 467 amino acids indicated a gene product of Mr 51,747. Transcription of glnA occurred from a C residue located 32 bases upstream of an ATG considered to be the initiator codon because (i) it had a nearby potential ribosome-binding site and (ii) an open reading frame translated from this site indicated good N-terminal homology to 10 other procaryotic GSs. Sequences similar to the consensus RNA polymerase recognition sites at -10 and -35 were present at the appropriate distance upstream of the transcription initiation site. As expected from earlier genetic studies indicating that expression of A. vinelandii glnA did not depend on the rpoN (ntrA; sigma 54) gene product, no sigma 54 recognition sequences were present, nor was there significant regulation of glnA expression by fixed nitrogen. Repeated attempts to construct glutamine auxotrophs by recombination of glnA insertion mutations were unsuccessful, Although the mutated DNA could be found by hybridization experiments in drug-resistant A. vinelandii transformants, the wild-type glnA region was always present. These results suggest that glnA mutations are lethal in A. vinelandii. In [14C]glutamine uptake experiments, very little glutamine was incorporated into cells, suggesting that glutamine auxotrophs are nonviable because they cannot be supplied with sufficient glutamine to support growth.
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PMID:Molecular analysis of the Azotobacter vinelandii glnA gene encoding glutamine synthetase. 197 37

The product of the rpoN gene is an alternative sigma factor of RNA polymerase which is required for transcription of a number of genes in members of the family Enterobacteriaceae, including those that specify enzymes of nitrogen assimilation, amino acid uptake, and degradation of a variety of organic molecules. We have previously shown that transcription of the pilin gene of Pseudomonas aeruginosa also requires RpoN (K. S. Ishimoto and S. Lory, Proc. Natl. Acad. Sci. USA 86:1954-1957, 1989) and have undertaken a more extensive survey of genes under RpoN control. Strains of P. aeruginosa that carry an insertionally inactivated rpoN gene were constructed and shown to be nonmotile because of the inability of these mutants to synthesize flagellin. The mutation in rpoN had no effect on expression of extracellular polypeptides, outer membrane proteins, and the alginate capsule. However, the rpoN mutants were glutamine auxotrophs and were defective in glutamine synthetase, indicating defects in nitrogen assimilation. In addition, the P. aeruginosa rpoN mutants were defective in urease activity. These findings indicate that the sigma factor encoded by the rpoN gene is used by P. aeruginosa for transcription of a diverse set of genes that specify biosynthetic enzymes, degradative enzymes, and surface components. These rpoN-controlled genes include pili and flagella which are required for full virulence of the organism.
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PMID:The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. 215 9

Growth of cells of Escherichia coli in nitrogen-limited medium induces the formation of glutamine synthetase, product of the glnA gene, and of other proteins that facilitate the assimilation of nitrogen-containing compounds. Transcription from the glnAp2 promoter of the glnALG operon requires the phosphorylation of nitrogen regulator I (NRI) and, for optimal transcription, the binding of NRI-phosphate to two sites that can be over 1,000 base pairs from the binding site for RNA polymerase. In other procaryotic genes, placement of an activator-binding site further upstream from the start site of transcription diminishes expression. To determine how NRI-phosphate activates transcription and why NRI-dependent transcription differs from activation in other systems, we constructed recombinant plasmids with small alterations between the binding sites for NRI-phosphate and RNA polymerase and between the two high-affinity NRI-binding sites. We demonstrate that tightly bound NRI-phosphate activated transcription from either side of the DNA helix when at least 30 base pairs separated NRI-phosphate from RNA polymerase. In contrast, activation from a partial NRI-binding site was effective only from one side of the DNA. We also observed that glnA expression was optimal when the two high-affinity NRI-binding sites were on the same side of the DNA helix. We explain these results on the basis of a hypothesis that a contact between RNA polymerase and NRI-phosphate bound to an upstream site determines the rate of glnA transcription.
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PMID:Activation of glnA transcription by nitrogen regulator I (NRI)-phosphate in Escherichia coli: evidence for a long-range physical interaction between NRI-phosphate and RNA polymerase. 257 9

The transcription of glnA, the structural gene for glutamine synthetase in enteric bacteria, is regulated by the phosphorylation and dephosphorylation of an effector protein, NRI. In its phosphorylated form the effector activates the initiation of transcription at promoters specific of sigma 54, rather than the abundant sigma 70. The ability of NRI-phosphate to stimulate the formation of open promoter-sigma 54 RNA polymerase complexes is enhanced by specific binding sites, located in the case of glnA 100 and 130 base pairs upstream from the transcriptional start site. These sites can be moved more than 1000 base pairs upstream or downstream without losing their effectiveness. The phosphorylation and dephosphorylation of NRI-NRI-phosphate is catalyzed by the modulator protein NRII. Its activity is controlled by an intracellular signal, the ratio of glutamine to 2-ketoglutarate, which is generated by glutamine synthetase in response to the environmental stimulus, the availability or lack of ammonia. The signal is transduced to the modulator by means of 2 additional proteins: uridylytransferase and PII.
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PMID:Regulation of transcription of the glnALG operon of Escherichia coli by protein phosphorylation. 257 99

Escherichia coli rpoN mutants lack sigma 54 and are therefore unable to initiate the transcription of glnA at glnAp2, which is required for the production of a high intracellular concentration of glutamine synthetase. We have found that the dependence on sigma 54 can be overcome by mutations that have apparently created a new sigma 70-dependent promoter. The position -35 RNA polymerase contact site of this new promoter overlaps glnAp2. The initiation of transcription at the new promoter is inhibited by sigma 54-RNA polymerase even in the absence of nitrogen regulator I-phosphate, the activator required for the initiation of transcription at glnAp2. The results suggest that in cells growing with an excess of nitrogen and therefore lacking nitrogen regulator I-phosphate, sigma 54-RNA polymerase is bound at glnAp2.
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PMID:Mutations that create new promoters suppress the sigma 54 dependence of glnA transcription in Escherichia coli. 288 48

The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.
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PMID:Sequence of the Bacillus subtilis glutamine synthetase gene region. 290 11

In enteric bacteria the products of two nitrogen regulatory genes, ntrA and ntrC, activate transcription of glnA, the structural gene encoding glutamine synthetase, both in vivo and in vitro. The ntrC product (gpntrC) is a DNA-binding protein, which binds to five sites in the glnA promoter-regulatory region and appears to activate transcription initiation. Using as an assay the stimulation of glnA transcription in a coupled in vitro transcription-translation system, we have partially purified the ntrA gene product (gpntrA). The following evidence is consistent with the view that gpntrA is a sigma subunit for RNA polymerase: (i) The gpntrA activity copurifies with the sigma 70 holoenzyme (E sigma 70) and core (E) forms of RNA polymerase through several steps but can be separated from them by chromatography on heparin agarose. (ii) After further purification by molecular sieve chromatography, the partially purified gpntrA fraction allows transcription of glnA from the same startpoint used in vivo; transcription is dependent on gpntrC and on added E. The gpntrA fraction does not allow transcription from promoters that we have used as controls, including lacUV5. E sigma 70 has the reverse specificity.
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PMID:Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. 299 66

We have developed two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria. The first procedure, based upon differential sedimentation, depends upon the association of GS with deoxyribonucleic acid in cell extracts. The second procedure, derived from the method of C. Gross et al (J. Bacteriol. 128:382-389, 1976) for purifying ribonucleic acid polymerase by polyethylene glycol (PEG) precipitation, enabled us to obtain high yields of GS from either small or large quantities of cells. We used the PEG procedure to purify GS from Klebsiella aerogenes, K. pneumoniae, Escherichia coli, Salmonella typhimurium, Rhizobium sp. strain 32H1, R. meliloti, Azotobacter vinelandii, Pseudomonas putida, Caulobacter crescentus, and Rhodopseudomonas capsulata. The purity of the GS obtained, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was high, and in many instances only a single protein band was detected.
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PMID:Purification of glutamine synthetase from a variety of bacteria. 610 84


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