The transduction of the nitrogen regulation signal in Saccharomyces cerevisiae.

In cells of Saccharomyces cerevisiae, using ammonia as a source of nitrogen, Gln3p is sequestered in the cytoplasm by Ure2p but enters the nucleus when the cells are shifted to a nonpreferred source of nitrogen such as proline. The interpretation of recently published observations provides evidence for the view that Ure2p is the sensor for a drop in the intracellular concentration of glutamine, a signal that results in the polyubiquitination of the vesicle responsible for retaining the Gln3p-Ure2p complex in the cytoplasm. As a consequence of the drop in glutamine concentration, Gln3p is able to enter the nucleus and to activate the transcription of nitrogen-regulated genes.

Nitrogen regulation in Saccharomyces cerevisiae.

Yeast cells can respond to growth on relatively poor nitrogen sources by increasing expression of the enzymes for the synthesis of glutamate and glutamine and by increasing the activities of permeases responsible for the uptake of amino acids for use as a source of nitrogen. These general responses to the quality of nitrogen source in the growth medium are collectively termed nitrogen regulation. In this review, we discuss the historical foundations of the study of nitrogen regulation as well as the current understanding of the regulatory networks that underlie nitrogen regulation.

DNA bending and the initiation of transcription at sigma54-dependent bacterial promoters.

We have examined the effects on transcription initiation of promoter and enhancer strength and of the curvature of the DNA separating these entities on wild-type and mutated enhancer-promoter regions at the Escherichia coli sigma54-dependent promoters glnAp2 and glnHp2 on supercoiled and linear DNA. Our results, together with previously reported observations by other investigators, show that the initiation of transcription on linear DNA requires a single intrinsic or induced bend in the DNA, as well as a promoter with high affinity for sigma54-RNA polymerase, but on supercoiled DNA requires either such a bend or a high affinity promoter but not both. The examination of the DNA sequence of all nif gene activator- or nitrogen regulator I-sigma54 promoters reveals that those lacking a binding site for the integration host factor have an intrinsic single bend in the DNA separating enhancer from promoter.

Role of GATA factor Nil2p in nitrogen regulation of gene expression in Saccharomyces cerevisiae.

We have identified the product of the NIL2 gene of Saccharomyces cerevisiae which contains a zinc finger region highly homologous to those of the GATA factors Gln3p and Nil1p as an antagonist of Nil1p and to a lesser extent of Gln3p. The expression of many nitrogen-regulated genes of Saccharomyces cerevisiae requires activation by GATA factor Gln3p or Nil1p and is prevented by the presence of glutamine in the growth medium. Disruption of NIL2 results in a great increase in the expression of NIL1 and of GAP1, the structural gene for the general amino acid permease, in glutamine-grown cells in response to activation by Nil1p.

Asparaginase II of Saccharomyces cerevisiae. GLN3/URE2 regulation of a periplasmic enzyme.

The production of some extracellular enzymes is known to be negatively affected by readily metabolized nitrogen sources such as NH4+ although there is no consensus regarding the involved mechanisms. Asparaginase II is a periplasmic enzyme of Saccharomyces cerevisiae encoded by the ASP3 gene. The enzyme activity is not found in cells grown in either ammonia, glutamine, or glutamate, but it is found in cells that have been subjected to nitrogen starvation or have been grown on a poor source of nitrogen such as proline.

Activation of transcription at sigma 54-dependent promoters on linear templates requires intrinsic or induced bending of the DNA.

The initiation of transcription (open complex formation) on supercoiled DNA templates carrying the sigma 54-dependent promoters glnAp2 or glnHp2 can be readily activated by NR1-phosphate bound to sites located 100 bp upstream from the transcriptional start site. In the case of glnAp2, open complex formation can also be activated by NR1-phosphate on a linear template, but in the case of glnHp2 activation on a linear template requires in addition to NR1-phosphate, a DNA-bending protein such as the histone-like protein HU or integration host factor (IHF). Moving the binding sites for NR1 200 bp further away from glnHp2 allows transcription to be activated equally well in the absence or presence of HU, and in this case IHF inhibits the open complex formation.

Interaction of the GATA factor Gln3p with the nitrogen regulator Ure2p in Saccharomyces cerevisiae.

We used cells carrying plasmids causing the overproduction of Gln3p, Ure2p, or both of these proteins to elucidate the ability of Ure2p to prevent the activation of gene expression by Gln3p in cells growing in a glutamine-containing medium. Our results indicate that Ure2p probably does not interfere with the binding of the GATA factor Gln3p to GATAAG sites but acts directly on Gln3p to block its ability to activate transcription.

Two transcription factors, Gln3p and Nil1p, use the same GATAAG sites to activate the expression of GAP1 of Saccharomyces cerevisiae.

We present an analysis of the DNA region located upstream of GAP1, the structural gene for the general amino acid permease, which contains the sites required for activation of transcription of this gene in response to the nitrogen source of the growth medium. This gene is not expressed in media containing glutamine, and its transcription is activated in response to Gln3p in cells using glutamate as the source of nitrogen and by Nil1p in cells using urea as the source of nitrogen. We show that full response to both activators requires the presence of two GATAAG sites, as well as the presence of auxiliary sites located in the interval between 602 and 453 bp from the translational start site.

Role of the GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated genes.

We have isolated the NIL1 gene, whose product is an activator of the transcription of nitrogen-regulated genes, by virtue of the homology of its zinc-finger domain to that of the previously identified activator, the product of GLN3. Disruption of the chromosomal NIL1 gene enabled us to compare the effects of Gln3p and of Nil1p on the expression of the nitrogen-regulated genes GLN1, GDH2, and GAP1, coding respectively for glutamine synthetase, NAD-linked glutamate dehydrogenase, and general amino acid permease. Our results show that the nature of GATAAG sequence that serve as the upstream activation sequence elements for these genes determines their abilities to respond to Gln3p and Nil1p.

Recognition of nitrogen-responsive upstream activation sequences of Saccharomyces cerevisiae by the product of the GLN3 gene.

We describe the purification of the product of the GLN3 gene of Saccharomyces cerevisiae and the demonstration that the purified product, Gln3p, binds specifically to the DNA sequences GATAAG and GATTAG, previously identified as nitrogen-responsive upstream activation sequences (UASN). When Gln3p is overproduced, it is released from the cells in a highly aggregated form incapable of specific binding to UASN. We used Gln3p tagged with six histidine codons at the 5' terminus and equipped with a galactose-inducible promoter to overproduce histidine-tagged Gln3p.

A sequence-induced superhelical DNA segment serves as transcriptional enhancer.

The initiation of transcription at the sigma 54-dependent promoter glnAp2 of Escherichia coli is activated by the protein NR1(NTRC)-phosphate, which binds to two sites located upstream of the promoter that together constitute an enhancer. The cooperative binding facilitates the oligomerization of NR1-phosphate endowing it with the ATPase activity required for its ability to serve as transcriptional activator. We show here that these sites can be replaced by sequence-dependent superhelical inserts, lacking any homology to the nucleotide sequence of the enhancers.

Activation of the dephosphorylation of nitrogen regulator I-phosphate of Escherichia coli.

The transcription of sigma 54 RNA polymerase-dependent nitrogen-regulated genes is activated by nitrogen regulator I (NRI)-phosphate. The kinase NRII is responsible for the phosphorylation of NRI. It has been shown that NRII also has the ability to dephosphorylate NRI-phosphate but only when PII is present at a concentration greatly in excess of that of NRII.

Transcriptional and posttranslational regulation of the general amino acid permease of Saccharomyces cerevisiae.

The cellular level and activity of the general amino acid permease, the product of the GAP1 gene of Saccharomyces cerevisiae, are regulated at the level of transcription by two systems, the products of URE2/GLN3 and NIL1 in response to the nitrogen sources of the growth medium and inactivation in response to the presence of glutamine or glutamate. Active permease is phosphorylated. The addition of glutamine causes rapid dephosphorylation and inactivation of the permease with the same kinetics, which is followed by slower disappearance of the protein.

A charmed life.

Boris Magasanik was born in Kharkoff, Ukraine, on December 19, 1919. He received his preliminary and secondary education in Vienna, Austria, and studied chemistry at the University of Vienna in 1937. He continued his studies at City College, New York (BS, 1941) and after one semester of graduate study at Pennsylvania State University, served in the US Army in England and France from 1942-1945.

The glnB region of the Escherichia coli chromosome.

We present sequences of the glnB gene of Escherichia coli and of two open reading frames (ORFs) located directly upstream of glnB and transcribed in the same direction. The major transcriptional start sites for glnB are located between ORF-2 and glnB, but some transcription of glnB is initiated at the promoter for ORF-1. The putative amino acid sequence of the ORF-2 product has high homology to that of response regulators which by phosphorylation acquire the ability to activate transcription of sigma 54-dependent promoters.

Positive and negative effects of DNA bending on activation of transcription from a distant site.

Transcription of the Escherichia coli glnHPQ operon, which encodes components of the high-affinity glutamine transport system, is activated by nitrogen regulator I (NRI)-phosphate in response to nitrogen limitation. NRI-phosphate binds to sites upstream from the sigma 54-dependent glnHp2 promoter and activates transcription by catalyzing the isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex. On linear DNA, the initiation of glnHp2 transcription requires in addition to NRI-phosphate the presence of integration host factor (IHF), which binds to a site located between the NRI-binding sites and the promoter.

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.

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.

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.