Interaction of negative (CytT) and positive (cAMP-CRP) regulation in the promoter region of the uridine phosphorylase (udp) gene in Escherichia coli K-12

Interaction of negative (CytR) and positive (cAMP-CRP) control in the promoter region of the uridine phosphorylase (udp) gene of Escherichia coli has been studied by using udp-lac operon fusions in which the structural lacZ gene is expressed from the wild type promoter udpP+ or from mutant promoters udpP1 and udpP18. The specific activity of beta-galactosidase was examined in these fusions in cytR+ and cytR- backgrounds after introduction of specific mutations in crp locus, crp* and crp(a) altering interaction of CRP protein with catabolite-sensitive promoters. The data obtained using crp* mutation confirm the proposed model of the udp gene regulation, according to which CytR repressor protein interferes with CRP binding site in the promoter-operator region of the udp gene and thereby prevents the positive action of cAMP-CRP complex on the udp expression. Additional data in favor of this model were obtained using crp(a) mutation which most probably alters the structure of CRP protein in such a way that it exhibits more high affinity to the udp promoter, as compared to the CytR repressor protein. Indeed, taken by itself, the crp(a) mutation did not lead to any increase in the expression of udpP+-lac fusion under the conditions of cAMP limitation (on glucose-grown cells), in spite of whether or not the CytR repressor was present. However, when combined with the ptsG mutation or when cells were grown on succinate medium, complete constitutive expression of udpP+-lac fusion is observed, even in the presence of the cytR gene product. The effect of the crp(a) mutation was virtually the same in strains harboring udpP1-lac fusion. These data are in accordance with suggestion that udpP1 is a mutation in the site of the promoter-operator region that responds to the cytR gene product, while the corresponding binding site for CRP protein is still unaltered in this mutant. On the other hand, the crp(a) mutation causes only slight alteration in the expression of udpP18-lac fusion, providing additional evidence that udpP18 mutation seems to comprise a modification of the promoter-operator region, where binding sites for CRP and CytR proteins overlap.     

Mutations in the promoter regions of the malEFG and malK-lamB operons of Escherichia coli K12

The malB region of Escherichia coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes. Expression of the malB operons is under the positive control of the malT gene product (MalT) and maltose and of the crp gene product (CRP) and cyclic AMP. Strains in which the lac genes have been fused to malE or malK are unable to use lactose as carbon source if they have been deleted for malT or crp. Mutations in the malB region allowing such fusion strains to grow on lactose have been isolated. These and previously isolated mutations were genetically characterized. As regards the malEp promoter mutations, malEp9, malEp1 and malEp6 create new promoters that are MalT and CRP independent. malEp9 and malEp1 change residues -1 and -2, respectively, of malEp without altering its activity. malEp6 duplicates six base-pairs between residues -22 and -23. malEp3 improves the -10 region hexamer. malEp5 deletes residues -29 to -62. It creates a new promoter that is MalT independent, CRP dependent, likely by fusing together functional regions of malEp that are normally apart. malEp5 also reduces the expression of malK-lamB, suggesting the existence of a link between the malEp and malKp promoters. As regards the malKp mutations, malKp6 changes residue -81 of malKp without altering its activity. It creates a new promoter, which is MalT independent, CRP dependent, likely by using a pre-existing cyclic AMP/CRP binding site. malKp102 changes residue -36, two bases upstream of the -35 region hexamer. It decreases the activity of malKp by at least four orders of magnitude and likely alters the MalT binding site. These results are discussed in terms of regulatory interactions within the malB control region.     

Synergistic activation of the human adrenomedullin gene promoter by Sp1 and AP-2alpha

Adrenomedullin (AM) is a potent vasodilator peptide, which is ubiquitously expressed and has various biological actions, such as proliferative action and anti-oxidative stress action. AM expression is induced by various stresses, such as hypoxia and inflammatory cytokines, and during cell differentiation. The human AM gene promoter region (-70/-29) contains binding sites for stimulatory protein 1 (Sp1) and activator protein-2alpha (AP-2alpha), and has been shown to be important for the AM gene expression during cell differentiation to macrophages or adipocytes. We here show that Sp1 and AP-2alpha synergistically activate the AM gene promoter. Co-transfection of the reporter plasmid containing the AM promoter region (-103/-29) with Sp1 and AP-2alpha expression plasmids showed that Sp1 and AP-2alpha synergistically increased the promoter activity in HeLa cells. Sp1 or AP-2alpha alone caused only small increases in the promoter activity. EMSA showed that Sp1 bound to the promoter region (-70/-29), whereas AP-2alpha bound to a more upstream promoter region (-103/-71). Thus, the synergistic activation of the human AM gene promoter by Sp1 and AP-2alpha may be mediated by the binding of Sp1 to the promoter region (-70/-29) and the interaction with AP-2alpha, which binds to the promoter region (-103/-71).     

Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP

CRP—cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene ( crp* ). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of β-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of β-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS—PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp * mRNA levels. The level of CRP* protein correlates with β-galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp * expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya⁻ crp * cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose.     

Genomic organization and chromosomal location of the mouse vasoactive intestinal polypeptide 1 (VPAC1) receptor.

The gene encoding the mouse vasoactive intestinal polypeptide type 1 (VPAC1) receptor was cloned, and its structural organization was determined. The gene (Vipr1) is more than 16 kb in length and is divided into 13 exons. The 5'-flanking region is highly GC-rich and lacks an apparent TATA box, but contains a CCAAT box, three potential Sp1-binding sites, and two potential AP-2-binding sites. Promoter analysis of the 5'-flanking region of Vipr1 using a luciferase gene reporter system revealed that the isolated 5'-flanking region has functional promoter activity. The mouse Vipr1 gene is encoded by a single gene, which was mapped to the distal region of mouse chromosome 9. This region is syntenic with human chromosome 3p, where the human VPAC1 receptor gene has been mapped.