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B Magasanik

Publications and source records attributed to B Magasanik.

At least 55 records · Page 3Linked to original sources
PubMed1988

Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli.

It has previously been shown that phosphorylated nitrogen regulator I (NRI-phosphate) is the activator responsible for increasing the transcription of glnA, the structural gene for glutamine synthetase, and that NRII catalyzes the transfer of the gamma-phosphate of ATP to NRI. We have now shown that the reaction of ATP with NRII results in the reversible transfer of the gamma-phosphate of ATP to a histidine residue of NRII. In turn, NRII-phosphate transfers its phosphate reversibly to an aspartic residue of NRI. NRI-phosphate is hydrolyzed to NRI and inorganic phosphate in a divalent cation-requiring autocatalytic reaction.

Adenosine Diphosphate↗
PubMed1988

Regulation of nitrogen assimilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes.

Mutations in the GLN3 gene prevented a normal increase in the NAD-glutamate dehydrogenase and glutamine synthetase levels in glutamate-grown Saccharomyces cerevisiae cells, whereas mutations in the URE2 gene resulted in high levels of these enzymes in glumate- and glutamine-grown cells. A ure2 gln3 double mutant had low levels of glutamate dehydrogenase and glutamine synthetase in cells grown on glutamate and glutamine; thus, gln3 mutations were epistatic to the ure2 mutations. The results suggest that the GLN3 product is capable of promoting increases in enzyme levels in the absence of a functional URE2 product and that the URE2 product antagonizes the GLN3 product. The URE2 and GLN3 genes were also found to regulate the level of arginase activity. This regulation is completely independent of the regulation of arginase by substrate induction. The activities of glutamate dehydrogenase, glutamine synthetase, and arginase were higher in cells grown on glutamate as the nitrogen source than they were in cells grown under a nitrogen-limiting condition. It had previously been shown that the levels of these enzymes can be increased by glutamine deprivation. We propose that the URE2-GLN3 system regulates enzyme synthesis, in response to glutamine and glutamate, to adjust the intracellular concentration of ammonia so as to maintain glutamine at the level required for optimal growth.

Ammonia↗
PubMed1987

Initiation of transcription at the bacterial glnAp2 promoter by purified E. coli components is facilitated by enhancers.

The initiation of transcription from the nitrogen-regulated promoter glnAp2 requires RNA polymerase containing sigma 54, the transcriptional activator NRI, and the protein kinase NRII, responsible for the conversion of NRI to the active NRI-phosphate. NRI-phosphate does not increase the ability of sigma 54-containing RNA polymerase to bind to the promoter, but rather stimulates the conversion of an initial promoter:polymerase complex to the transcriptionally active open complex. The presence on the DNA template of high-affinity binding sites for NRI/NRI-phosphate, normally located 130 and 100 bp upstream of the site of transcription initiation, results in a 4- to 5-fold lowering of the concentration of NRI required for the formation of the open complex. These high-affinity NRI binding sites facilitate open complex formation when they are moved to positions 700 bp further upstream or 950 bp downstream of glnAp2 on linear DNA templates.

Base Sequence↗
PubMed1987

Mutations that create new promoters suppress the sigma 54 dependence of glnA transcription in Escherichia coli.

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.

Base Sequence↗
PubMed1986

Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter.

Transcription of the Escherichia coli glnALG operon, whose products are glutamine synthetase and the regulatory proteins NRII and NRI, is activated by nitrogen deprivation. Initiation of transcription at the nitrogen-regulated promoter glnAp2 requires sigma 60, the product of rpoN (glnF, ntrA), and NRI, the product of glnG (ntrC). We have now shown that the ability of this promoter to be activated by a low intracellular concentration of NRI depends on two binding sites for NRI located approximately 110 and 140 bp, respectively, upstream of the start of transcription. Moving these binding sites more than 1000 bp does not diminish the ability of NRI to stimulate transcription at glnAp2. Thus, the NRI binding sites resemble enhancers in eukaryotic cells.

Binding Sites↗
PubMed1986

Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli.

Transcription from nitrogen-regulated promoters, such as glnAp2, requires the glnG gene product, NRI, as well as the rpoN(glnF) gene product, sigma60, and is regulated by the glnL gene product, NRII. We find that in a reaction mixture containing NRI, NRII, and ATP, NRII catalyzes the transfer of the gamma phosphate of ATP to NRI. This covalent modification of NRI occurs concurrently with the acquisition of the ability by the reaction mixture to activate transcription from glnAp2. In the presence of PII, the product of glnB, NRII catalyzes the removal of the phosphate from NRI-phosphate. This reaction occurs concurrently with the loss by the reaction mixture of the ability to activate transcription from glnAp2. On the basis of this evidence, we propose that NRI-phosphate activates transcription from nitrogen-regulated promoters and that the role of NRII is control of the formation and breakdown of NRI-phosphate in response to cellular signals of nitrogen availability.

Escherichia coli↗
PubMed1985

Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG, and glnL.

We have shown that the purified glnF (ntrA) product of Escherichia coli binds to core RNA polymerase. Together these proteins initiated transcription at the nitrogen-regulated promoter glnAp2 on a supercoiled template. The initiation of transcription at glnAp2 on a linear template required in addition NRI, the product of glnG (ntrC), and NRII2302, the product of a mutant allele of glnL (ntrB). These results identify the glnF product as a new sigma factor specifically required for the transcription of nitrogen-regulated and of nitrogen-fixation promoters. We propose rpoN as the proper designation for glnF, and sigma 60 for its product. Our results indicate that sigma 60 RNA polymerase recognizes the nitrogen-regulated/nitrogen-fixation promoter consensus sequence C-T-G-G-Y-A-Y-R-N4-T-T-G-C-A. Initiation of transcription in the intact cell appears to require in addition the active form of NRI, the product of glnG. Conversion of NRI to its active form is apparently brought about by NRII, the product of glnL, in response to nitrogen deprivation.

DNA, Bacterial↗
PubMed1985

Expression of glnA in Escherichia coli is regulated at tandem promoters.

We have determined that the glnA gene of the complex glnALG operon of Escherichia coli is transcribed from tandem promoters. Expression from the upstream promoter, glnAp1, requires the catabolite activating protein, is repressed by nitrogen regulator I (NRI), the product of glnG, and produces a transcript with an untranslated leader of 187 nucleotides. Expression from the downstream promoter, glnAp2, requires NRI as well as the glnF product; full expression also requires growth in a nitrogen-limited environment. The downstream transcript has an untranslated leader of 73 nucleotides. We also provide evidence that the function of the glnL product is to mediate the interconversion of NRI between a form capable of activating glnAp2 and an inactive form in response to changes in the intracellular concentration of ammonia. The function of the two minor promoters of the glnALG operon, glnAp1 and glnLp, is to maintain the products of glnA, glutamine synthetase, an essential enzyme, and of glnG, NRI, an activator of nitrogen-controlled genes, during carbon-limited growth.

Base Sequence↗
PubMed1985

Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli.

We have isolated insertion and deletion mutants in glnB, the structural gene of PII, a member of the adenylylation system for glutamine synthetase of Escherichia coli, to study the role of PII in the regulation of the synthesis of glutamine synthetase and of histidase in response to nitrogen deprivation or excess. We have studied the effects of this mutation alone and combined with null mutations resulting from the insertion of transposons or from a deletion in the other genes affecting this regulation, glnD, glnF (ntrA), glnG (ntrC), and glnL (ntrB). Our results confirm that only the products of glnF and glnG are essential for this regulation. In cells of the wild type, the response is mediated by the products of glnD and glnB via the product of glnL. In the condition of nitrogen excess, PII, the product of glnB, appears to convert the product of glnL to a form that prevents the activation of transcription of the structural genes for glutamine synthetase and for histidase by the products of glnF and glnG. During nitrogen deprivation, uridylyltransferase, the product of glnD, is activated by the intracellular excess of 2-ketoglutarate over glutamine and converts PII to PII-UMP and changes the form of the glnL product to one that stimulates the activation of transcription of glutamine synthetase and histidase by the products of glnF and glnG.

Bacterial Proteins↗
PubMed1984

Biochemical and physiological aspects of glutamine synthetase inactivation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae glutamine synthetase is inactivated in vivo by the addition of glutamine or ammonia. Inactivation is characterized by a specific loss of synthetase activity; transferase activity remains stable. Several physiological perturbations cause inactivation, such as carbon starvation or limitation for a required amino acid, which could cause a buildup of glutamine. The kinetics of reappearance of synthetase activity after inactivation suggest that the process is reversible in vivo. No change in the native size of the enzyme was associated with inactivation but there appears to be a change in the immunological properties of the enzyme subunit.

Ammonia↗
PubMed1984

Isolation of Bacillus subtilis mutants pleiotropically insensitive to glucose catabolite repression.

A pleiotropic mutant of Bacillus subtilis was isolated which overproduced in the presence of glucose several enzymes whose synthesis is subject to glucose catabolite repression. Examination of intracellular metabolites suggested that the mutation may have resulted in a defect in glycolysis, increasing phosphoenolpyruvate and decreasing pyruvate, 2-ketoglutarate, and oxaloacetate.

Aconitate Hydratase↗
PubMed1984

2-Ketoglutarate and the regulation of aconitase and histidase formation in Bacillus subtilis.

In contrast to wild-type cells, the Bacillus subtilis mutant SF109 that lacks the active 2-ketoglutarate dehydrogenase enzymatic complex is unable to increase the specific activity of two enzymes subject to glucose catabolite repression, aconitase and histidase, during limitation of growth by glucose. Examination of the intracellular metabolite pools in the mutant and wild-type cells grown in excess and limiting glucose medium showed that the complete derepression of aconitase and histidase could be correlated with the decrease in the intracellular concentration of 2-ketoglutarate. The complete repression of aconitase that occurred in wild-type and mutant cells could be correlated with a high intracellular concentration of 2-ketoglutarate.

Aconitate Hydratase↗
PubMed1984

Synthesis of oxaloacetate in Bacillus subtilis mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex.

Bacillus subtilis mutants deficient in the 2-ketoglutarate dehydrogenase enzymatic complex required aspartate for growth at wild-type rates on carbon sources for which synthesis of the degradative enzymes is sensitive to catabolite repression (e.g., poor carbon sources), but did not require aspartate for growth on carbon sources which exert catabolite repression (e.g., good carbon sources). Measurement of metabolite pools in a mutant lacking the 2-ketoglutarate dehydrogenase active complex showed that the aspartate requirement for growth on poor carbon sources resulted from a deficiency in intracellular oxaloacetate pools even through pyruvate carboxylase was present at levels corresponding to those in wild-type cells. The oxaloacetate deficiency most likely resulted from the inability of the mutant to regenerate oxaloacetate from citrate due to the enzymatic block in the tricarboxylic acid cycle. Mutants in the enzymes of the dicarboxylic acid half of the citric acid cycle similarly required aspartate for wild-type growth in minimal medium. These results suggested that the complete turning of the tricarboxylic acid cycle is involved in the maintainance of oxaloacetate levels in B. subtilis. The ability of the mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex to grow at wild-type rates on media containing good carbon sources in the absence of exogenous aspartate is not understood.

Asparagine↗
PubMed1984

Identification and regulation of the glnL operator-promoter of the complex glnALG operon of Escherichia coli.

We sequenced the 274-nucleotide intercistronic glnA-glnL region of Escherichia coli to localize regulatory regions postulated from genetic evidence. The transcriptional start of glnLp, identified by S1 nuclease mapping, preceded the structural gene by 32 bases. NR1, the glnG gene product and a repressor of glnLp, protected from DNase digestion a region of DNA between -12 and +15 from the transcriptional start. A mutation rendering glnLp insensitive to NRI was within the protected region in a-TGCA- sequence found in all nitrogen-regulated operons, providing evidence for involvement of this sequence in interactions with NRI. We also observed in the intercistronic region a potential rho-independent terminator preceding glnLp and a sequence previously found in other intercistronic regions.

Amino Acid Sequence↗
PubMed1984

Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae.

Mutants of the yeast Saccharomyces cerevisiae have been isolated which fail to derepress glutamine synthetase upon glutamine limitation. The mutations define a single nuclear gene, GLN3, which is located on chromosome 5 near HOM3 and HIS1 and is unlinked to the structural gene for glutamine synthetase, GLN1. The three gln3 mutations are recessive, and one is amber suppressible, indicating that the GLN3 product is a positive regulator of glutamine synthetase expression. Four polypeptides, in addition to the glutamine synthetase subunit are synthesized at elevated rates when GLN3+ cultures are shifted from glutamine to glutamate media as determined by pulse-labeling and one- and two-dimensional gel electrophoresis. The response of all four proteins is blocked by gln3 mutations. In addition, the elevated NAD-dependent glutamate dehydrogenase activity normally found in glutamate-grown cells is not found in gln3 mutants. Glutamine limitation of gln1 structural mutants has the opposite effect, causing elevated levels of NAD-dependent glutamate dehydrogenase even in the presence of ammonia. We suggest that there is a regulatory circuit that responds to glutamine availability through the GLN3 product.

Ammonia↗
PubMed1984

Three regulatory systems control production of glutamine synthetase in Saccharomyces cerevisiae.

Production of glutamine synthetase in Saccharomyces cerevisiae is controlled by three regulatory systems. One system responds to glutamine levels and depends on the positively acting GLN3 product. This system mediates derepression of glutamine synthetase in response to pyrimidine limitation as well, but genetic evidence argues that this is an indirect effect of depletion of the glutamine pool. The second system is general amino acid control, which couples derepression of a variety of biosynthetic enzymes to starvation for many single amino acids. This system operates through the positive regulatory element GCN4. Expression of histidinol dehydrogenase, which is under general control, is not stimulated by glutamine limitation. A third system responds to purine limitation. No specific regulatory element has been identified, but depression of glutamine synthetase is observed during purine starvation in gln3 gcn4 double mutants. This demonstrates that a separate purine regulatory element must exist. Pulse-labeling and immunoprecipitation experiments indicate that all three systems control glutamine synthetase at the level of subunit synthesis.

Amino Acids↗