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Biomedical subjects

B Magasanik

Publications and source records attributed to B Magasanik.

At least 37 records · Page 2Linked to original sources
PubMed1992

Phosphorylation of nitrogen regulator I of Escherichia coli induces strong cooperative binding to DNA essential for activation of transcription.

We studied the effect of phosphorylation of nitrogen regulator I (NRI) on its binding properties. Both phosphorylated and unphosphorylated NRI bind linearly to a single binding site but cooperatively to two adjacent binding sites. Cooperative binding of NRI is severely affected by phosphorylation: half-maximal binding of NRI-phosphate is at 20-fold lower concentrations than that of unphosphorylated NRI. This is more due to a huge increase in the cooperativity constant--which is the strength of interaction between two NRI dimers--than to an increase in the microscopic binding constant which is the binding affinity to a single binding site. In vitro transcription and DNA footprinting experiments showed that occupation of a single binding site by NRI is not enough for efficient activation and that activation only occurs at a higher NRI concentration. We propose an activation mechanism for NRI in which the phosphorylation of NRI induces a conformational change in the N-terminal domains of the NRI-phosphate dimers, which now interact strongly with each other, leading to a tetramerization of NRI upon binding to two adjacent binding sites. We propose that not the phosphorylation of NRI itself but rather the tetramerization of NRI-phosphate on DNA binding induces the conformational change of the central domain to the active conformation.

Bacterial Proteins↗
PubMed1992

Sequence of the GLN1 gene of Saccharomyces cerevisiae: role of the upstream region in regulation of glutamine synthetase expression.

The GLN1 gene, encoding glutamine synthetase in Saccharomyces cerevisiae, was sequenced, and its encoded polypeptide was shown to have significant homology to other eukaryotic glutamine synthetases. S1 analysis has defined the transcriptional start site of the gene. Upstream analysis of the gene using lacZ fusions has verified transcriptional control of the gene and has identified a nitrogen upstream activation sequence which is required for the increased transcription of GLN1 seen when glutamine is replaced by glutamate as the nitrogen source. cis-acting sites required for the increased transcription in response to purine starvation also have been localized.

Amino Acid Sequence↗
PubMed1991

Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli.

The glnHPQ operon of Escherichia coli encodes components of the high-affinity glutamine transport system. One of the two promoters of this operon, glnHp2, is responsible for expression of the operon under nitrogen-limiting conditions. The general nitrogen regulatory protein (NRI) binds to two overlapping sites centered at -109 and -122 from the transcription start site and, when phosphorylated, activates transcription of glnHp2 by catalyzing isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex. The DNA-bending protein integration host factor (IHF) binds to a site immediately upstream of glnHp2 and enhances the activation of open complex formation by NRI phosphate. The NRI-binding sites can be moved several hundred base pairs further upstream without altering the ability of NRI phosphate to activate open complex formation. We propose that the IHF-induced bend can facilitate or obstruct the interaction between NRI phosphate and the closed complex depending on the relative positions of NRI phosphate and sigma 54-RNA polymerase on the DNA.

Bacterial Proteins↗
PubMed1991

Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain.

The GLN3 gene of Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen. We cloned the GLN3 gene and constructed null alleles by gene disruption. GLN3 is not essential for growth, but increased copies of GLN3 lead to a drastic decrease in growth rate. The complete nucleotide sequence of the GLN3 gene was determined, revealing one open reading frame encoding a polypeptide of 730 amino acids, with a molecular weight of approximately 80,000. The GLN3 protein contains a single putative Cys2/Cys2 zinc finger which has homology to the Neurospora crassa NIT2 protein, the Aspergillus nidulans AREA protein, and the erythroid-specific transcription factor GATA-1. Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLN1, the gene encoding glutamine synthetase. Neither control of transcription nor control of initiation of translation of GLN3 is important for regulation in response to glutamine availability.

Alleles↗
PubMed1991

Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae.

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.

Amino Acid Sequence↗
PubMed1991

The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to the nitrogen source and has homology to glutathione s-transferases.

The URE2 gene of Saccharomyces cerevisiae has been cloned and sequenced. It encodes a predicted polypeptide of 354 amino acids with a molecular weight of 40,226. Deletion of the first 63 amino acids does not have any effect on the function of the protein. Studies with disruption alleles of the URE2 and GLN3 genes showed that both genes regulate GLN1 and GDH2, the structural genes for glutamine synthetase and NAD-linked glutamate dehydrogenase, respectively, at the transcriptional level, but expression of the regulatory genes does not appear to be regulated. Active URE2 gene product was required for the inactivation of glutamine synthetase upon addition of glutamine to cells growing with glutamate as the source of nitrogen. The predicted URE2 gene product has homology to glutathione S-transferases. The gene has been mapped to chromosome XIV, 5.9 map units from petX and 3.4 map units from kex2.

Amino Acid Sequence↗
PubMed1991

Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae.

We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.

Blotting, Northern↗
PubMed1991

Control site location and transcriptional regulation in Escherichia coli.

The regulatory regions for 119 Escherichia coli promoters have been analyzed, and the locations of the regulatory sites have been cataloged. The following observations emerge. (i) More than 95% of promoters are coregulated with at least one other promoter. (ii) Virtually all sigma 70 promoters contain at least one regulatory site in a proximal position, touching at least position -65 with respect to the start point of transcription. There are not yet clear examples of upstream regulation in the absence of a proximal site. (iii) Operators within regulons appear in very variable proximal positions. By contrast, the proximal activation sites of regulons are much more fixed. (iv) There is a forbidden zone for activation elements downstream from approximately position -20 with respect to the start of transcription. By contrast, operators can occur throughout the proximal region. When activation elements appear in the forbidden zone, they repress. These latter examples usually involve autoregulation. (v) Approximately 40% of repressible promoters contain operator duplications. These occur either in certain regulons where duplication appears to be a requirement for repressor action or in promoters subject to complex regulation. (vi) Remote operator duplications occur in approximately 10% of repressible promoters. They generally appear when a multiple promoter region is coregulated by cyclic AMP receptor protein. (vii) Sigma 54 promoters do not require proximal or precisely positioned activator elements and are not generally subject to negative regulation. Rationales are presented for all of the above observations.

Escherichia coli↗
PubMed1990

Role of the promoter in activation of transcription by nitrogen regulator I phosphate in Escherichia coli.

The protein nitrogen regulator I (NRI)-phosphate is known to activate the initiation of transcription of the Escherichia coli glnA gene. This activation is facilitated by the binding of the protein to NRI-specific sites located upstream of the sigma 54-dependent glnA promoter. To determine whether binding of NRI-phosphate to upstream sites is sufficient for activation, we placed several promoters not normally activated by NRI-phosphate downstream of NRI binding sites and measured activation in intact cells and in an in vitro transcription system. We found that the sigma 70-dependent lac promoter was not activated, that the sigma 54-dependent Klebsiella pneumoniae nifH promoter was weakly activated, and that a nifH promoter altered in the RNA polymerase binding site was almost as well activated as the glnA promoter. We conclude that the sensitivity of the susceptible promoter depends on the presence of NRI binding sites, but that the presence of bound NRI-phosphate upstream of a promoter is not sufficient for activation of transcription by RNA polymerase. This activation is determined by the structure of the RNA polymerase binding site. We suggest that sigma 54-but not sigma 70-dependent promoters are susceptible to activation by NRI-phosphate and that the nucleotide sequence of the sigma 54-RNA polymerase binding site is an important determinant of the efficiency of activation.

Bacterial Proteins↗
PubMed1990

Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae.

We cloned GDH2, the gene that encodes the NAD-linked glutamate dehydrogenase in the yeast Saccharomyces cerevisiae, by purifying the enzyme, making polyclonal antibodies to it, and using the antibodies to screen a lambda gt11 yeast genomic library. A yeast strain with a deletion-disruption allele of GDH2 which replaced the wild-type gene grew very poorly with glutamate as a nitrogen source, but growth improved significantly when the strain was also provided with adenine or other nitrogenous compounds whose biosynthesis requires glutamine. Our results indicate that the NAD-linked glutamate dehydrogenase catalyzes the major, but not sole, pathway for generation of ammonia from glutamate. We also isolated yeast mutants that lacked glutamate synthase activity and present evidence which shows that normally NAD-linked glutamate dehydrogenase is not involved in glutamate biosynthesis, but that if the enzyme is overexpressed, it may function reversibly in intact cells.

Alleles↗
PubMed1989

Three regulatory systems control expression of glutamine synthetase in Saccharomyces cerevisiae at the level of transcription.

The GLN1 gene of Saccharomyces cerevisiae was cloned by complementation of a gln1 auxotroph. A GLN1-lacZ fusion was constructed to assay GLN1 promoter activity. beta-Galactosidase and glutamine synthetase expression in chromosomally integrated GLN1-lacZ fusion strains were co-regulated in response to a shift from glutamine to glutamate as the nitrogen source, purine limitation, and 3-aminotriazole-induced histidine starvation. Regulation of GLN1 expression by each of the three pathways occurred at the transcriptional level. Increased accumulation of GLN1 mRNA was observed within 5 min after a shift from glutamine to glutamate as the nitrogen source. After 5 min, GLN1 mRNA levels were constant. The level of GLN1 transcript was reduced by approximately 75% within 5 min following glutamine addition to the cells growing with glutamate as nitrogen source. This indicates that the GLN1 message is unstable and has a half-life of approximately 3 min. Deletion analysis indicated that the sequences required for GLN1 expression are located within approximately 350 bp upstream from the transcriptional initiation site.

Chromosome Deletion↗
PubMed1989

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.

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.

Bacterial Proteins↗
PubMed1989

Mutations in the glnG gene of Escherichia coli that result in increased activity of nitrogen regulator I.

Mutations in the glnG gene of Escherichia coli that result in increased activity of nitrogen regulator I (NRI), the product of glnG, were obtained by two different selection procedures. The mutant proteins were purified and characterized. The concentrations of mutant proteins needed to activate transcription at the glnAp2 promoter were three to four times lower than that of the wild-type NRI. The rate of phosphorylation of these proteins and the stability of mutant NRI phosphate were found to be similar to those of the wild-type NRI. In one of the mutants, the site of the mutation was localized in the DNA region specifying the central domain of NRI.

Bacterial Proteins↗
PubMed1989

Gene regulation from sites near and far.

Although regulation of gene expression from distant sites would seem to have many advantages, this strategy is utilized far more frequently in eukaryotic cells than in prokaryotic cells. The inherent flexibility of DNA allows factors bound to distant regulatory sites to interact with transcription initiation factors or RNA polymerase components at the promoter of the regulated gene. Regulatory domains that are closely linked to the promoter lose this spatial flexibility, and must lie within a defined region in the vicinity of the promoter. The major drawback to regulation of gene expression from distant sites is that any gene with a promoter located within the effective radius of the regulatory factor will respond to its control. Thus, promoters of independently regulated genes must be separated by ample amounts of nonspecific DNA. Prokaryotic cells may have evolved to favor regulation of transcription from closely linked sites because this mechanism allows them to eliminate excess DNA, which may in turn enable them to achieve rapid growth in an energy-limited environment.

DNA↗
PubMed1988

Research on bacteria in the mainstream of biology.

The study of the genetics, biochemistry, and physiology of bacteria during the last 40 years has provided the concepts and methods for the study of cells of all types at the molecular level. Although much is already known about the mechanisms bacteria use to regulate the expression of their genes, a great deal more remains to be discovered that will have relevance to both prokaryotic and eukaryotic cells. Similarly, the study in bacteria of the transactions of DNA, of the synthesis and function of the cell membrane, of differentiation, and of the interaction with eukaryotic cells will undoubtedly produce results of general importance. The advantages of using bacteria for these studies include their simple noncompartmented structure, the accessibility of their genetic material, and the possibility of correlating the expression of a gene in the intact cell with its expression in a system composed of highly purified components. Finally, the comparative study of a wide variety of microorganisms may result in a better understanding of the evolution of prokaryotes and eukaryotes and lead to a comprehensive theory of cell biology.

Bacteria↗
PubMed1988

Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism.

We demonstrate by using purified bacterial components that the protein kinases that regulate chemotaxis and transcription of nitrogen-regulated genes, CheA and NRII, respectively, have cross-specificities: CheA can phosphorylate the Ntr transcription factor NRI and thereby activate transcription from the nitrogen-regulated glnA promoter, and NRII can phosphorylate CheY. In addition, we find that a high intracellular concentration of a highly active mutant form of NRII can suppress the smooth-swimming phenotype of a cheA mutant. These results argue strongly that sensory transduction in the Ntr and Che systems involves a common protein phosphotransfer mechanism.

Bacterial Proteins↗