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 GLNI 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.
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.In Saccharomyces cerevisiae, the expression of a number of genes whose products enable the organism to use a variety of compounds as sources of nitrogen is activated by the product of the GLN3 gene, Gln3p. This zinc finger protein exerts its effect by binding to GATAAG sequences located upstream of the regulated genes (1). The product of another gene, URE2, prevents the activation of the regulated genes by Gln3p in cells growing with glutamine as the source of nitrogen (5, 6).We have cloned the GLN3 and URE2 genes on plasmids, enabling us to overproduce the products of these genes (4, 7). We now present the results of experiments with cells that overproduce either one or both of the products of these genes. Our experiments suggest that Ure2p acts directly on Gln3p, converting it to a form that is unable to activate transcription.As previously reported, overproduction of Gln3p results in very slow growth, with a mass doubling time of more than 10 h in minimal media (7). We examined the effects of Ure2p on the growth inhibition exerted by Gln3p. To this end, we transformed strain PM38 (MAT␣ leu2-3,112 ura3-52) with plasmid pPM49 (2 m GAL10-GLN3 [7]). We also transformed this strain with plasmids carrying the URE2 gene fused to GAL10 as well as the LEU2 gene in order to create strains that overproduce both Gln3p and Ure2p. The high-copy-number plasmid pLE-8 was constructed by inserting a 1.7-kb StuI-SalI fragment of p8 containing the GAL10-URE2 fusion into the BamHI site of YEp13 (2, 3). The resulting plasmid has a LEU2 selectable marker and the GAL10 upstream activating sequence driving the URE2 gene. The single-copy centromere plasmid pLC-8 (YCpGAL10-URE2) was constructed by replacing the URA3 fragment of the p8 plasmid with the SalIXhoI LEU2-containing fragment of YEp13.The results presented in Fig. 1 show that overproduction of Gln3p in the strain carrying the GLN3 gene fused to GAL10 causes strong growth inhibition and that growth is restored by the overproduction of Ure2p resulting from the simultaneous presence of the plasmid carrying URE2 fused to GAL10.The effect of Ure2p is also demonstrated by the ability of Ure2p to depress the ability of overproduced Gln3p to activate the synthesis of glutamine synthetase. As shown in Table 1, overproduction of Gln3p enables the cell to produce glutamine synthetase during growth with glutamine as the source of nitrogen, but this increase in the level of the enzyme is largely prevented by the simultaneous overproduction of Ure2p.We have previously shown that Ure2p blocks neither the FIG. 1. Suppression of GLN3 lethality by overexpression of URE2...
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.
The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.
Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits α, β and γ that in Thauera Aromatica T1 strain are encoded by the tutD, tutG and tutF genes respectively. The large α-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small β- and γ-subunits has remained unclear. We have over-expressed all three subunits of benzylsuccinate synthase in E. coli, both individually and in combination. Co-expression of the γ-subunit (but not the β-subunit) is essential for efficient expression of the α-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an α2β2γ2 hexamer by nickel-affinity chromatography through a ‘His6’ affinity tag engineered onto the C-terminus of the α-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the β-subunit and the other with the γ-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly a mutant γ-subunit lacking the [4Fe-4S] cluster is unable to stabilize the α-subunit when the proteins are co-expressed.
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