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B Magasanik

Publications and source records attributed to B Magasanik.

At least 19 recordsLinked to original sources
PubMed1997

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.

Bacterial Proteins↗
PubMed1997

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. In this report it is shown that the formation of this enzyme is dependent upon the functional GLN3 gene and that the response to nitrogen availability is under the control of the URE2 gene product. In this respect the expression of ASP3 is similar to the system that regulates the GLN1, GDH2, GAP1, and PUT4 genes that codes for glutamine synthetase, NAD-linked glutamate dehydrogenase, general amino-acid permease, and high affinity proline permease, respectively.

Asparaginase↗
PubMed1997

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. The primary effect of the elimination of Nil2p appears to be an increase in the intracellular level of Nil1p, which in turn is responsible for increased expression of GAP1. Experiments using an artificial UAS (upstream activating site) consisting of three GATAAGATAAG sites revealed that Nil2p exerts its effect by competing primarily with Nil1p and less effectively with Gln3p for these sites. Apparently, the principal role of Nil2p is to prevent activation of transcription by Nil1p unless Nil1p has been converted to a more active state by the absence of glutamine and glutamate.

DNA-Binding Proteins↗
PubMed1996

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. Furthermore, replacement of the DNA segment separating the binding sites for NR1 from glnAp2 by a random sequence of bases of equal length, does not reduce open complex formation on supercoiled DNA but prevents open complex formation on linear DNA unless HU is provided. These observations indicate that with binding sites for NR1 located in their usual position, 100 bp from the transcriptional start site, the DNA segment separating these sites from the promoter must be either intrinsically bent or bent by HU or, in the case of glnHp2, by IHF to allow contact between activator and the sigma 54-RNA polymerase-promoter complex. Computer simulation of the shape of the DNA suggests that in the case of glnAp2, but not of glnHp2 or the altered glnAp2, this segment has an intrinsic curvature of 70 degrees.

Bacterial Proteins↗
PubMed1996

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.

Base Sequence↗
PubMed1996

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. The fact that both Gln3p and Nil1p utilize GATAAG sites to activate transcription is reflected in the high homology of the zinc finger regions of the two proteins.

Amino Acid Transport Systems↗
PubMed1995

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. The results further indicate that Gln3p is inactivated by an increase in the intracellular concentration of glutamine and that Nil1p is inactivated by an increase in intracellular glutamate.

Amino Acid Sequence↗
PubMed1995

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. These superhelical inserts, irrespective of their chirality, are as effective as the paired sites in binding NR1-phosphate and in stimulating its oligomerization. We conclude that a specific sequence of nucleotides and the three-dimensional structure of DNA can determine its affinity for the NR1 activator protein capable of binding to DNA.

Adenosine Triphosphatases↗
PubMed1995

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. These results suggest that inactivation of the permease results from its dephosphorylation.

Amino Acid Transport Systems↗
PubMed1995

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. The material was denatured, adsorbed to an Ni-nitrilotriacetic acid (NTA)-agarose column, eluted with imidazole, and after renaturation further purified on a gel filtration column. We then demonstrated the specific binding of the more than 90% pure Gln3p to the UASN by gel shift and footprinting methods.

Base Sequence↗
PubMed1995

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. We have now shown that glutamate enables PII to stimulate the dephosphorylation of NRI-phosphate when present in equimolar concentration with NRII. This effect of glutamate appears to be a backup control that becomes effective when the normal regulation of PII activity is disabled.

Bacterial Proteins↗
PubMed1994

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. He obtained his PhD degree in biochemistry from Columbia University in 1948. A faculty member in the Department of Bacteriology and Immunology at Harvard Medical School from 1951 to 1960, he then continued his career as Professor of Microbiology at MIT where he served as head of the Department from 1967 to 1977. He became a member of the National Academy of Sciences USA in 1969 and received the Selman A Waksman Award in 1993. He is currently the Jacques Monod Professor Emeritus at MIT and is the author of over 240 publications dealing with microbial physiology and the regulation of gene expression in bacteria and yeast.

Biochemistry↗
PubMed1993

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. The product of ORF-1 showed no similarity to other known proteins. The product of neither ORF-1 nor ORF-2 is necessary for the ability of PII, the product of glnB, to bring about the repression of glutamine synthetase in response to nitrogen excess. On the other hand, the product of hmpA, a gene located on the other side of glnB and transcribed in the opposite direction, appears to play an auxiliary role in this process.

Amino Acid Sequence↗
PubMed1992

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. On supercoiled DNA, IHF does not play an essential role, but enhances the activation of transcription by NRI-phosphate. We found that at a mutant glnHp2 promoter with increased affinity for sigma 54-RNA polymerase, the initiation of transcription can be activated equally well by NRI-phosphate in the presence or absence of IHF. Binding of IHF to its site does not increase the binding of sigma 54-RNA polymerase to the glnHp2 promoter; instead, our data suggest that IHF bends the DNA to align the activator with the closed sigma 54-RNA polymerase promoter complex to facilitate the interactions that result in open complex formation. In the absence of IHF, NRI-phosphate can activate transcription whether its binding sites are on the same face of the DNA helix as the sigma 54-RNA polymerase or on the opposite face. IHF enhances transcription when the three proteins are located on the same face of the helix, but strongly inhibits transcription when any one of the proteins is located on the opposite face.

Amino Acid Transport Systems, Basic↗