RESUMEN
Saccharomyces cerevisiae activates a regulatory network called "general control" that provides the cell with sufficient amounts of protein precursors during amino acid starvation. We investigated how starvation for nitrogen affects the general control regulatory system, because amino acid biosynthesis is part of nitrogen metabolism. Amino acid limitation results in the synthesis of the central transcription factor Gcn4p, which binds to specific DNA-binding motif sequences called Gcn4-protein-responsive elements (GCREs) that are present in the promoter regions of its target genes. Nitrogen starvation increases GCN4 transcription but efficiently represses expression of both a synthetic GCRE6::lacZ reporter gene and the natural amino acid biosynthetic gene ARO4. Repression of Gcn4p-regulated transcription by nitrogen starvation is independent of the ammonium sensing systems that include Mep2p and Gpa2p or Ure2p and Gln3p but depends on the four upstream open reading frames in the GCN4 mRNA leader sequence. Efficient translation of GCN4 mRNA is completely blocked by nitrogen starvation, even when cells are simultaneously starved for amino acids and eukaryotic initiation factor-2 alpha is fully phosphorylated by Gcn2p. Our data suggest that nitrogen starvation regulates translation of GCN4 by a novel mechanism that involves the four upstream open reading frames but that still acts independently of eukaryotic initiation factor-2 alpha phosphorylation by Gcn2p.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Proteínas Quinasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Nitrógeno/metabolismo , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.
Asunto(s)
Genes Esenciales/genética , Morfogénesis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , División Celular , Polaridad Celular , Quitina/metabolismo , Epistasis Genética , Genes Fúngicos/genética , Haploidia , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Nitrógeno/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genéticaRESUMEN
Diploid strains of the budding yeast Saccharomyces cerevisiae change the pattern of cell division from bipolar to unipolar when switching growth from the unicellular yeast form (YF) to filamentous, pseudohyphal (PH) cells in response to nitrogen starvation. The functions of two transmembrane proteins, Bud8p and Bud9p, in regulating YF and PH cell polarity were investigated. Bud8p is highly concentrated at the distal pole of both YF and PH cells, where it directs initiation of cell division. Asymmetric localization of Bud8p is independent of the Rsr1p/Bud1p GTPase. rsr1/bud1 mutations are epistatic to bud8 mutations, placing Rsr1p/Bud1p downstream of Bud8p. In YF cells, Bud9p is also localized at the distal pole, yet deletion of BUD9 favours distal bud initiation. In PH cells, nutritional starvation for nitrogen efficiently prevents distal localization of Bud9p. Because Bud8p and Bud9p proteins associate in vivo, we propose Bud8p as a landmark for bud initiation at the distal cell pole, where Bud9p acts as inhibitor. In response to nitrogen starvation, asymmetric localization of Bud9p is averted, favouring Bud8p-mediated cell division at the distal pole.
Asunto(s)
Proteínas Fúngicas/fisiología , Glicoproteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Polaridad Celular , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fúngicas/análisis , Proteínas Fúngicas/genética , Genotipo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Modelos Biológicos , Plásmidos , Proteínas Recombinantes de Fusión/análisis , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/genéticaRESUMEN
The two highly conserved RAS genes of the budding yeast Saccharomyces cerevisiae are redundant for viability. Here we show that haploid invasive growth development depends on RAS2 but not RAS1. Ras1p is not sufficiently expressed to induce invasive growth. Ras2p activates invasive growth using either of two downstream signaling pathways, the filamentation MAPK (Cdc42p/Ste20p/MAPK) cascade or the cAMP-dependent protein kinase (Cyr1p/cAMP/PKA) pathway. This signal branch point can be uncoupled in cells expressing Ras2p mutant proteins that carry amino acid substitutions in the adenylyl cyclase interaction domain and therefore activate invasive growth solely dependent on the MAPK cascade. Both Ras2p-controlled signaling pathways stimulate expression of the filamentation response element-driven reporter gene depending on the transcription factors Ste12p and Tec1p, indicating a crosstalk between the MAPK and the cAMP signaling pathways in haploid cells during invasive growth.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , AMP Cíclico/metabolismo , FMN Reductasa , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas ras/genética , Proteínas ras/metabolismo , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Secuencia Conservada , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Genes Supresores , Haploidia , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Datos de Secuencia Molecular , Mutación , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiaeRESUMEN
The CPC2 gene of the budding yeast Saccharomyces cerevisiae encodes a G beta-like WD protein which is involved in regulating the activity of the general control activator Gcn4p. The CPC2 gene encodes a premRNA which is spliced and constitutively expressed in the presence or absence of amino acids. Loss of CPC2 gene function suppresses a deletion of the GCN2 gene encoding the general control sensor kinase, but not a deletion in the GCN4 gene. The resulting phenotype has resistance against amino-acid analogues. The Neurospora crassa cpc-2 and the rat RACK1 genes are homologues of CPC2 that complement the yeast cpc2 deletion. The cpc2 delta mutation leads to increased transcription of Gcn4p-dependent genes under non-starvation conditions without increasing GCN4 expression or the DNA binding activity of Gcn4p. Cpc2p-mediated transcriptional repression requires the Gcn4p transcriptional activator and a Gcn4p recognition element in the target promoter. Frameshift mutations resulting in a shortened G beta-like protein cause a different phenotype that has sensitivity against amino-acid analogues similar to a gcn2 deletion. Cpc2p seems to be part of an additional control of Gcn4p activity, independent of its translational regulation.
Asunto(s)
Aminoácidos/fisiología , Proteínas de Arabidopsis , Citocromos c , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Proteínas Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Acetatos/farmacología , Actinas/genética , Amitrol (Herbicida)/farmacología , Secuencia de Bases , Northern Blotting , Western Blotting , Grupo Citocromo c/genética , Epistasis Genética , Mutación del Sistema de Lectura , Regulación Fúngica de la Expresión Génica , Genotipo , Glucosa/farmacología , Crecimiento , Hidroliasas/genética , Isocitrato Deshidrogenasa/análisis , Operón Lac , Datos de Secuencia Molecular , Neurospora/genética , Proteínas Quinasas/genética , Factores de Tiempo , beta-Galactosidasa/análisisRESUMEN
In Saccharomyces cerevisiae the GCN4 gene encodes the transcriptional activator of the "general control" system of amino acid bioynthesis, a network of at least 12 different biosynthetic pathways. We characterized the consequences of the general control response upon the signal "amino acid starvation" induced by the histidine analogue 3-aminotriazole with respect to Gcn4p levels in more detail. Therefore, we established test systems to monitor the time course of different parameters, including GCN4 mRNA, Gcn4 protein, Gcn4p DNA binding activity, as well as Gcn4p transactivation ability. We observed a biphasic response of Gcn4p activity in the cell. At first, translation of GCN4 mRNA is induced within 20 min after switch to starvation conditions. However, an additional increase in GCN4 transcript steady state level was observed, leading to an additional second phase of GCN4 expression after 3-4 h of starvation. The DNA binding activity of Gcn4p, as well as the ability to activate transcription of target genes, correlate with the amount of Gcn4 protein in the cell, suggesting that under the tested conditions there is no additional regulation of DNA binding or transactivation ability of Gcn4p, respectively.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Amitrol (Herbicida)/farmacología , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Genes Reporteros , Histidina/metabolismo , Sistemas de Lectura Abierta , Proteínas Quinasas/genética , Transducción de Señal , Transcripción GenéticaRESUMEN
The small GTP-binding protein Ras and heterotrimeric G-proteins are key regulators of growth and development in eukaryotic cells. In mammalian cells, Ras functions to regulate the mitogen-activated protein kinase pathway in response to growth factors, whereas many heterotrimeric GTP-binding protein alpha-subunits modulate cAMP levels through adenylyl cyclase as a consequence of hormonal action. In contrast, in the yeast Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins that regulate adenylyl cyclase. Of the two yeast G-protein alpha-subunits (GPA1 and GPA2), only GPA1 has been well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation. In this report, we show that deletion of the GPA2 gene encoding the other yeast G-protein alpha-subunit leads to a defect in pseudohyphal development. Also, the GPA2 gene is indispensable for normal growth in the absence of Ras2p. Both of these phenotypes can be rescued by deletion of the PDE2 gene product, which inactivates cAMP by cleavage, suggesting that these phenotypes can be attributed to low levels of intracellular cAMP. In support of this notion, addition of exogenous cAMP to the growth media was also sufficient to rescue the phenotype of a GPA2 deletion strain. Taken together, our results directly demonstrate that a G-protein alpha-subunit can regulate the growth and pseudohyphal development of S. cerevisiae via a cAMP-dependent mechanism. Heterologous expression of mammalian G-protein alpha-subunits in these yeast GPA2 deletion strains could provide a valuable tool for the mutational analysis of mammalian G-protein function in an in vivo null setting.
Asunto(s)
AMP Cíclico/fisiología , Proteínas Fúngicas/fisiología , Proteínas de Unión al GTP/fisiología , Saccharomyces cerevisiae/fisiología , AMP Cíclico/análisisRESUMEN
14-3-3 proteins are highly conserved ubiquitous proteins whose explicit functions have remained elusive. Here, we show that the S. cerevisiae 14-3-3 homologs BMH1 and BMH2 are not essential for viability or mating MAPK cascade signaling, but they are essential for pseudohyphal-development MAPK cascade signaling and other processes. Activated alleles of RAS2 and CDC42 induce pseudohyphal development and FG(TyA)-lacZ signaling in Bmh+ strains but not in ste20 (p65PAK) or bmh1 bmh2 mutant strains. Moreover, Bmh1p and Bmh2p associate with Ste20p in vivo. Three alleles of BMH1 encode proteins defective for FG(TyA)-lacZ signaling and association with Ste20p, yet these alleles complement other 14-3-3 functions. Therefore, the 14-3-3 proteins are specifically required for RAS/MAPK cascade signaling during pseudohyphal development in S. cerevisiae.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Transducción de Señal/fisiología , Proteínas ras/fisiología , Proteínas 14-3-3 , Adaptación Fisiológica , Alelos , Evolución Biológica , Supervivencia Celular/fisiología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/metabolismo , Glucógeno/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Operón Lac/genética , Quinasas Quinasa Quinasa PAM , Datos de Secuencia Molecular , Mutagénesis/fisiología , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Reproducción/fisiología , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestructura , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from growth as single yeast form (YF) cells to a multicellular form consisting of filaments of pseudohyphal (PH) cells. Filamentous growth is regulated by an evolutionarily conserved signaling pathway that includes the small GTP-binding proteins Ras2p and Cdc42p, the protein kinases Ste20p, Ste11p and Ste7p, and the transcription factor Ste12p. Here, we designed a genetic screen for mutant strains defective for filamentous growth (dfg) to identify novel targets of the filamentation signaling pathway, and we thereby identified 16 different genes, CDC39, STE12, TEC1, WHI3, NAB1, DBR1, CDC55, SRV2, TPM1, SPA2, BNI1, DFG5, DFG9, DFG10, BUD8 and DFG16, mutations that block filamentous growth. Phenotypic analysis of dfg mutant strains genetically dissects filamentous growth into the cellular processes of signal transduction, bud site selection, cell morphogenesis and invasive growth. Epistasis tests between dfg mutant alleles and dominant activated alleles of the RAS2 and STE11 genes, RAS2Val19 and STE11-4, respectively, identify putative targets for the filamentation signaling pathway. Several of the genes described here have homologues in filamentous fungi, where they also regulate fungal development.
Asunto(s)
Elementos Transponibles de ADN/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Genes Fúngicos , Haploidia , Datos de Secuencia Molecular , Mutagénesis , Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Transducción de Señal , Proteínas ras/metabolismo , Animales , GTP Fosfohidrolasas/metabolismo , Genotipo , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Mamíferos , Plásmidos , Proteínas Recombinantes/biosíntesis , Recombinación Genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , beta-Galactosidasa/biosíntesis , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiaeRESUMEN
Transcription from the yeast TRP4 promoter initiates at two basal (i127 and i76) and three GCN4 dependent (i31, i25 and i12) initiator elements. All of these elements contain not more than one deviation from the earlier proposed initiator consensus sequence PuPuPyPuPu, a pyrimidine nucleotide flanked on either side by two purine nucleotides. A point mutation analysis of these elements in various combinations was performed and revealed that the central pyrimidine nucleotide and at least one of the 3' flanking purine nucleotides of the PuPuPyPuPu consensus sequence are essential but alone not sufficient to define a functional initiator element. Multiple cryptic transcription start sites, which function independently whether they are located on the coding or the non-coding strand, can replace the function of mutated initiator elements and therefore the overall level of transcription initiation is not affected. The sequence specificity is identical for basal and GCN4 dependent initiator elements demonstrating that they are functionally homologous. These findings imply that the role of initiator elements is to 'focus' the start point(s) of transcription to distinct sites located in the region between the site(s) of the assembly of the transcriptional complex and the start codon of translation.
Asunto(s)
Antranilato Fosforribosiltransferasa/genética , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , ADN de Hongos , Datos de Secuencia Molecular , Mutagénesis Sitio-DirigidaRESUMEN
The yeast transcriptional regulator protein GCN4 harbors the bZIP DNA binding motif, which is common to a family of DNA-binding proteins in eukaryotic organisms from yeast to man. GCN4 and the mammalian activator protein AP-1 (jun/fos) regulate transcription by binding the same consensus DNA sequence ATGA (C/G)TCAT. GCN4 positively regulates the production of precursors of protein synthesis in yeast cells in response to the environmental signal "amino acid starvation." We find three GCN4 responsive elements (GCREs) in the 5'-flanking region of the purine biosynthetic gene ADE4 and demonstrate that GCN4 efficiently activates transcription of ADE4. Two GCREs are essential to synergistically activate ADE4 transcription by binding GCN4. The distal GCRE1 is also required for basal transcription of ADE4. Therefore, transcription factor GCN4 affects, in addition to protein biosynthesis, also nucleotide biosynthesis and, comparable to its mammalian counterpart AP-1, has a more general function within the yeast cell than previously assumed.
Asunto(s)
Proteínas Fúngicas/farmacología , Genes Fúngicos , Proteínas Quinasas , Nucleótidos de Purina/biosíntesis , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/farmacología , Activación Transcripcional , Secuencia de Bases , Northern Blotting , Proteínas de Unión al ADN/farmacología , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Nucleótidos de Purina/genética , ARN de Hongos/genética , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
The gene ARO7 encodes the monofunctional enzyme chorismate mutase, a branch point enzyme in the aromatic amino acid biosynthetic pathway in Saccharomyces cerevisiae. We investigated the transcription of the ARO7 gene. Three 5' ends at positions -36, -56 and -73 and the 3' end of the transcripts 146 bp downstream of the translational stop codon were mapped. As in the promoters of other aromatic amino acid biosynthetic genes, a recognition element for the GCN4 transcriptional activator of amino acid biosynthesis is located 425 base pairs (bp) upstream of the first transcriptional start point. This element binds GCN4 specifically in vitro. Northern analysis and determination of the specific enzyme activity reveals however, that the element is not sufficient to mediate transcriptional regulation by GCN4 in vivo. We thus suggest that in addition to a consensus sequence capable of binding the GCN4 protein other factors like, for example, chromatin structure, determine whether a recognition site for a transcription factor functions as an upstream activation sequence.
Asunto(s)
Corismato Mutasa/genética , Proteínas de Unión al ADN , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Northern Blotting , Southern Blotting , Corismato Mutasa/metabolismo , Mapeo Cromosómico , Cromosomas Fúngicos , Secuencia de Consenso , Desoxirribonucleasa I/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimologíaRESUMEN
The yeast TRP4 promoter contains three responsive elements (GCREs) for the 'general control' transcriptional activator GCN4, which are arranged in two upstream elements, UAS1 (GCRE1) and UAS2 (GCRE2 and GCRE3). A point mutation analysis of these elements revealed that all three GCREs are required for GCN4-dependent transcription, but none are involved in basal transcription. Basal transcription and GCN4-dependent transcription use completely different initiator elements in the TRP4 promoter. UAS1 acts synergistically with UAS2 to activate the GCN4-dependent transcription of TRP4. A consensus TATA box can functionally replace the UAS2 element to allow normal GCN4-dependent transcription, suggesting that UAS2 is analogous to the TATA element of other promoters. GCN4 might therefore activate transcription by exhibiting two alternative functions within the natural TRP4 promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Reguladores , Regiones Promotoras Genéticas , Proteínas Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Alelos , Secuencia de Bases , Northern Blotting , Desoxirribonucleasa I , Datos de Secuencia Molecular , Mutación , Mapeo Nucleótido , Poli A/análisis , Poli A/genética , ARN/análisis , ARN/genética , ARN MensajeroRESUMEN
Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of Mr 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by 1H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate with a Hill coefficient of 1.71 and a [S]0.5 value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an [S]0.5 value of 1.2 mM in the presence of 10 microM tryptophan and an increased [S]0.5 value of 8.6 mM in the presence of 300 microM tyrosine. In the mutant enzyme, a loss of cooperativity was observed, and [S]0.5 was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.
Asunto(s)
Corismato Mutasa/genética , Isomerasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sitios de Unión , Corismato Mutasa/aislamiento & purificación , Activación Enzimática , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Peso Molecular , Mutación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Two regulatory proteins, PHO2 and the general control regulator GCN4, bind in vitro to the promoter of the tryptophan biosynthetic TRP4 gene; the TRP4 gene product catalyses the phosphoribosylation of anthranilate. PHO2 binds specifically to the TRP4 promoter, but does not bind to any other TRP promoter. PHO2 and GCN4 proteins bind in a mutually exclusive manner to the same sequence, UAS1, one of two GCN4 binding sites in the TRP4 promoter. UAS1 is the major site for GCN4-dependent TRP4 activation. The second GCN4 binding site, UAS2, interacts with GCN4 alone. PHO2 binding interferes with the general control response of TRP4 under low phosphate conditions and simultaneous amino acid starvation and thus the PHO2 regulatory protein connects phosphate metabolism and amino acid biosynthesis in yeast. The GCN4 protein mediates the response of the transcriptional apparatus to the environmental signal 'amino acid limitation', while PHO2 seems to be the phosphate sensor that adjusts the response to the availability of phosphate precursors.
Asunto(s)
Genes Fúngicos , Saccharomyces cerevisiae/genética , Triptófano/genética , Secuencia de Bases , Sitios de Unión , Unión Competitiva , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The ARO3 gene encodes one of two 3-deoxy-D-arabino-heptulosonate-7-phosphate isoenzymes in Saccharomyces cerevisiae catalyzing the first step in the biosynthesis of aromatic amino acids. The ARO3-encoded 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) is feedback inhibited by phenylalanine; its isoenzyme, the ARO4 gene product, is inhibited by tyrosine. Both genes ARO3 and ARO4 are strongly regulated under the general control regulatory system. Cells carrying only one intact isogene are phenotypically indistinguishable from a wild-type strain when grown on minimal medium. The complete functional ARO3 promoter comprises 231 base pairs and contains only one TGACTA binding site for the general control activator protein GCN4. Mutating this element to TTACTA inhibits binding of GCN4 and results in a decreased basal level of ARO3 gene product and slow growth of a strain defective in its isogene ARO4. In addition, ARO3 gene expression cannot be elevated under amino acid starvation conditions. An ARO3 aro4 strain with gcn4 genetic background has the same phenotype of low ARO3 gene expression and slow growth. The amount of GCN4 protein present in repressed wild-type cells therefore seems to contribute to a basal level of ARO3 gene expression. The general control activator GCN4 has thus two functions: (i) to maintain a basal level of ARO3 transcription (basal control) in the presence of amino acids and (ii) to derepress the ARO3 gene to a higher transcription rate under amino acid starvation (general control).
Asunto(s)
3-Desoxi-7-Fosfoheptulonato Sintasa/biosíntesis , Aldehído-Liasas/biosíntesis , ADN Polimerasa I/biosíntesis , Proteínas Fúngicas/fisiología , Genes Fúngicos , Genes Reguladores , Regiones Promotoras Genéticas , Proteínas Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/fisiología , 3-Desoxi-7-Fosfoheptulonato Sintasa/genética , ADN/análisis , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Mutación , Plásmidos , Saccharomyces cerevisiae/enzimología , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The TRP4 gene of Saccharomyces cerevisiae, which encodes anthranilate phosphoribosyl transferase (E.C. 2.4.2.18), is subject to the general control of amino acid biosynthesis. The regulation takes place at the transcriptional level by increasing the amount of initiation and not by changing the stability of mRNA. We have observed a change in the utilization of TRP4 mRNA start sites, depending on whether cells were grown under repressing or derepressing conditions. The function of promoter elements has been tested by deletion analysis with a plasmid-encoded TRP4 gene. A routinely practicable method was used for copy-number calibration of plasmids based on 2 micron DNA. Promoter structures and spacing problems in the TRP4 promoter region are discussed.