RESUMO
In Saccharomyces cerevisiae, the ability to use proline as a nitrogen source requires the Put3p transcriptional regulator, which turns on the expression of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources. Changes in target gene expression occur through an alteration in activity of the DNA-bound Put3p, a member of the Zn(II)2Cys6 binuclear cluster family of proteins. Here, we report that the 'on' conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppress a put3 mutation leading to non-inducibility of the pathway. In addition, the presence of proline causes a conformational change in the Put3 protein detected by increased sensitivity to thrombin or V8 protease. These findings suggest that Put3p shifts from an inactive to an activate state via conformational changes.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Prolina/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transativadores/química , Transativadores/metabolismo , Sequência de Bases , Sítios de Ligação , DNA Fúngico/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Epitopos/genética , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Mutação , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Serina Endopeptidases/metabolismo , Trombina/metabolismo , Transativadores/genética , Fatores de TranscriçãoAssuntos
Elementos de DNA Transponíveis , Escherichia coli/genética , Mutagênese Insercional/métodos , Sitios de Sequências Rotuladas , Proteínas Virais , beta-Galactosidase/genética , Sequência de Bases , Escherichia coli/enzimologia , Biblioteca Gênica , Integrases/metabolismo , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/genética , beta-Galactosidase/análiseRESUMO
Two new flavanones (1 and 2) with antibacterial activity were isolated from the methanolic extract of the dried leaves of Physena madagascariensis using activity against Staphylococcus aureus to guide the isolation. A third flavonoid, a flavanone dimer linked by a methylene group (3) was also isolated and proved to be inactive. The structures of 1 and 2 were established primarily from NMR studies, while that of 3 required more extensive mass spectrometric analysis. All three flavanones had lavandulyl units in the limonene form. Flavanones 1 and 2 were active against several bacteria at concentrations as low as 4 microM.
Assuntos
Antibacterianos/isolamento & purificação , Flavanonas , Flavonoides/isolamento & purificação , Folhas de Planta/química , Plantas Medicinais/química , Antibacterianos/química , Antibacterianos/farmacologia , Cromatografia em Gel , Dicroísmo Circular , Flavonoides/química , Flavonoides/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Madagáscar , Espectroscopia de Ressonância Magnética , Rotação Ocular , Espectrofotometria Ultravioleta , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Árvores/químicaRESUMO
Proline can serve as a nitrogen source for the yeast Saccharomyces cerevisiae when preferred sources of nitrogen are absent from the growth medium. PUT3, the activator of the proline utilization pathway, is required for the transcription of the genes encoding the enzymes that convert proline to glutamate. PUT3 is a 979 amino acid protein that constitutively binds a short DNA sequence to the promoters of its target genes, but does not activate their expression in the absence of induction by proline and in the presence of preferred sources of nitrogen. To understand how PUT3 is converted from an inactive to an active state, a dissection of its functional domains has been undertaken. Biochemical and molecular tests, domain swapping experiments, and an analysis of activator-constitutive and activator-defective mutant proteins indicate that PUT3 is dimeric and activates transcription with its negatively charged carboxyterminus, which does not appear to contain a proline-responsive domain. A mutation in the conserved central domain found in many fungal activators interferes with activation without affecting DNA binding protein stability. Intragenic suppressors of the central domain mutation have been isolated and analyzed.
