RESUMEN
Mutations in the rifampicin (Rif)-binding site of RNA polymerase (RNAP) confer antibiotic resistance and often have global effects on transcription that compromise fitness and stress tolerance of resistant mutants. We suggested that the non-essential genome, through its impact on the bacterial transcription cycle, may represent an untapped source of targets for combination antimicrobial therapies. Using transposon sequencing, we carried out a genome-wide analysis of fitness cost in a clinically common rpoB H526Y mutant. We find that genes whose products enable increased transcription elongation rates compound the fitness costs of resistance whereas genes whose products function in cell wall synthesis and division mitigate it. We validate our findings by showing that the cell wall synthesis and division defects of rpoB H526Y result from an increased transcription elongation rate that is further exacerbated by the activity of the uracil salvage pathway and unresponsiveness of the mutant RNAP to the alarmone ppGpp. We applied our findings to identify drugs that inhibit more readily rpoB H526Y and other RifR alleles from the same phenotypic class. Thus, genome-wide analysis of fitness cost of antibiotic-resistant mutants should expedite the discovery of new combination therapies and delineate cellular pathways that underlie the molecular mechanisms of cost.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Rifampin/farmacología , Bacterias/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Farmacorresistencia Bacteriana , Genoma Bacteriano , Mutación , Transcripción GenéticaRESUMEN
TFIIB is essential for transcription initiation by RNA polymerase II. TFIIB also cross-links to terminator regions and is required for gene loops that juxtapose promoter-terminator elements in a transcription-dependent manner. The Saccharomyces cerevisiae sua7-1 mutation encodes an altered form of TFIIB (E62K) that is defective for both start site selection and gene looping. Here we report the isolation of an ssl2 mutant, encoding an altered form of TFIIH, as a suppressor of the cold-sensitive growth defect of the sua7-1 mutation. Ssl2 (Rad25) is orthologous to human XPB and is a member of the SF2 family of ATP-dependent DNA helicases. The ssl2 suppressor allele encodes an arginine replacement of the conserved histidine residue (H508R) located within the DEVH-containing helicase domain. In addition to suppressing the TFIIB E62K growth defect, Ssl2 H508R partially restores both normal start site selection and gene looping. Moreover, Ssl2, like TFIIB, associates with promoter and terminator regions, and the diminished association of TFIIB E62K with the PMA1 terminator is restored by the Ssl2 H508R suppressor. These results define a novel, functional interaction between TFIIB and Ssl2 that affects start site selection and gene looping.
Asunto(s)
ADN Helicasas/metabolismo , Subunidades de Proteína/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factor de Transcripción TFIIB/metabolismo , Factor de Transcripción TFIIH/metabolismo , Sitio de Iniciación de la Transcripción , Secuencia de Aminoácidos , Animales , ADN Helicasas/química , ADN Helicasas/genética , Genes Supresores , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Regiones Terminadoras Genéticas , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/genética , Transcripción Genética/genéticaRESUMEN
Gene loops are dynamic structures that juxtapose promoterterminator regions of Pol II-transcribed genes. Although first described in yeast, gene loops have now been identified in yeast and mammalian cells. Looping requires components of the transcription preinitiation complex, the pre-mRNA 30-end processing machinery, and subunits of the nuclear pore complex. Loop formation is transcription-dependent, but neither basal nor activated transcription requires looping. Rather, looping appears to affect cellular memory of recent transcriptional activity, enabling a more rapid response to subsequent stimuli. The nuclear pore has been implicated in both memory and looping. Our working model is that loops are formed and/or maintained at the nuclear pore to facilitate hand-off of Pol II form the terminator to the promoter, thereby bypassing Pol II recruitment as the rate-limiting step in reactivation of transcription. Involvement of the nuclear pore also suggests that looping might facilitate mRNA export to the cytoplasm. The technology now exists to test these ideas.
Asunto(s)
Expresión Génica , Conformación de Ácido Nucleico , Transcripción Genética , Activación Transcripcional , Cromosomas/metabolismo , ADN Polimerasa II/metabolismo , Genoma , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismoRESUMEN
DNA loops that juxtapose the promoter and terminator regions of RNA polymerase II-transcribed genes have been identified in yeast and mammalian cells. Loop formation is transcription-dependent and requires components of the pre-mRNA 3'-end processing machinery. Here we report that looping at the yeast GAL10 gene persists following a cycle of transcriptional activation and repression. Moreover, GAL10 and a GAL1p-SEN1 reporter undergo rapid reactivation kinetics following a cycle of activation and repression-a phenomenon defined as "transcriptional memory"-and this effect correlates with the persistence of looping. We propose that gene loops facilitate transcriptional memory in yeast.
Asunto(s)
ADN de Hongos/genética , Regulación Fúngica de la Expresión Génica , Conformación de Ácido Nucleico , Saccharomyces cerevisiae/fisiología , Proteínas de Unión al ADN , N-Metiltransferasa de Histona-Lisina , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factores de TranscripciónRESUMEN
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a reiterated heptad sequence (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) that plays a key role in the transcription cycle, coordinating the exchange of transcription and RNA processing factors. The structure of the CTD is flexible and undergoes conformational changes in response to serine phosphorylation and proline isomerization. Here we report that the Ess1 peptidyl prolyl isomerase functionally interacts with the transcription initiation factor TFIIB and with the Ssu72 CTD phosphatase and Pta1 components of the CPF 3'-end processing complex. The ess1(A144T) and ess1(H164R) mutants, initially described by Hanes and coworkers (Yeast 5:55-72, 1989), accumulate the pSer5 phosphorylated form of Pol II; confer phosphate, galactose, and inositol auxotrophies; and fail to activate PHO5, GAL10, and INO1 reporter genes. These mutants are also defective for transcription termination, but in vitro experiments indicate that this defect is not caused by altering the processing efficiency of the cleavage/polyadenylation machinery. Consistent with a role in initiation and termination, Ess1 associates with the promoter and terminator regions of the PMA1 and PHO5 genes. We propose that Ess1 facilitates pSer5-Pro6 dephosphorylation by generating the CTD structural conformation recognized by the Ssu72 phosphatase and that pSer5 dephosphorylation affects both early and late stages of the transcription cycle.
Asunto(s)
Isomerasa de Peptidilprolil/metabolismo , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Transcripción Genética , Proteínas Portadoras/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Modelos Biológicos , Mutación/genética , Peptidilprolil Isomerasa de Interacción con NIMA , Fosfoproteínas Fosfatasas , Fosfoserina/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Procesamiento de Término de ARN 3' , ARN Polimerasa II/química , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiones Terminadoras Genéticas , Factor de Transcripción TFIIB , Activación Transcripcional , Factores de Escisión y Poliadenilación de ARNm/metabolismoAsunto(s)
Células Eucariotas/fisiología , Transcripción Genética , Animales , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Humanos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Regiones Promotoras Genéticas , Conformación ProteicaRESUMEN
In an effort to identify novel components of the PHO regulon in Saccharomyces cerevisiae, we have isolated and characterized suppressors of the Pho(-) phenotype associated with deletion of the Pho4 transcriptional activator. Here we report that either a defective form of the Rsp5 E3 ubiquitin ligase or deletion of the End3 component of the endocytic pathway restores growth of the pho4 Delta mutant in the presence of limiting inorganic phosphate (P i). The spa1-1 suppressor allele of RSP5 encodes a phenylalanine-to-valine replacement at position 748 (F748V) within the catalytic HECT domain of Rsp5. Consistent with suppression due to impaired ubiquitin ligase activity, the heat-sensitive growth defect of the spa1-1 mutant is suppressed either by overexpression of ubiquitin or by osmotic stabilization. Western blot analyses revealed that the cellular levels of the Pho87 and Pho91 low affinity P i are markedly increased in the spa1-1 mutant, yet Pho84 high affinity P i transporter levels are unaffected. Furthermore, Pho87 and Pho91 are ubiquitinated in vivo in an Rsp5-dependent manner, and the Pho+ phenotype of the spa1-1 suppressor is dependent upon Pho87 and Pho91. We conclude that turnover of the low affinity P i transporters is initiated by Rsp5-mediated ubiquitination followed by internalization and degradation by the endocytic pathway.
Asunto(s)
Endocitosis/fisiología , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación/fisiología , Alelos , Sustitución de Aminoácidos , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Mutación Missense , Proteínas de Transporte de Fosfato/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
The ssu71 alleles of the TFG1 gene, which encodes the largest subunit of TFIIF, were isolated as suppressors of a TFIIB defect that affects the accuracy of transcription start site selection in the yeast Saccharomyces cerevisiae. Here we report that ssu71-1 also suppresses the cell growth and start site defects associated with an altered form of the Rpb1 subunit of RNA polymerase II (RNAP II). The ssu71-1 and ssu71-2 alleles were cloned and found to encode single amino acid replacements of glycine-363, either glycine to aspartic acid (G363D) or glycine to arginine (G363R). Two other charged replacements, G363E and G363K, were constructed by site-directed mutagenesis and suppress both TFIIB E62K and Rpb1 N445S, whereas neither G363A nor G363P exhibited any effect. G363 is phylogenetically conserved and its counterpart in human TFIIF (RAP74 G112) is located within the RAP74/RAP30 dimerization domain. We propose that the TFIIF dimerization domain is located in proximity to the B-finger of TFIIB near the active center of RNAP II where the TFIIB-TFIIF-RNAP II interface plays a key role in start site selection.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Factores de Transcripción TFII/química , Sitio de Iniciación de la Transcripción , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Dimerización , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Supresión Genética , Factor de Transcripción TFIIB/química , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismoRESUMEN
The phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II, consisting of ((1)YSPTSPS(7))(n) heptad repeats, encodes information about the state of the transcriptional apparatus that can be conveyed to factors that regulate mRNA synthesis and processing. Here we describe how the CTD code is read by two classes of protein phosphatases, plant CPLs and yeast Ssu72, that specifically dephosphorylate Ser(5) in vitro. The CPLs and Ssu72 recognize entirely different positional cues in the CTD primary structure. Whereas the CPLs rely on Tyr(1) and Pro(3) located on the upstream side of the Ser(5)-PO(4) target site, Ssu72 recognizes Thr(4) and Pro(6) flanking the target Ser(5)-PO(4) plus the downstream Tyr(1) residue of the adjacent heptad. We surmise that the reading of the CTD code does not obey uniform rules with respect to the location and phasing of specificity determinants. Thus, CTD code, like the CTD structure, is plastic.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Lisina/genética , Lisina/metabolismo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Escisión y Poliadenilación de ARNmRESUMEN
OBJECTIVES: Functional characterization of the erg1 mutant of ergosterol biosynthesis of Candida albicans. METHODS: We disrupted the ERG1 gene of C. albicans, which encodes squalene epoxidase (EC 1.14.99.7). Since the disruption of both alleles of ERG1 was lethal, the second allele of a heterozygous disruptant was placed under the control of a regulable promoter, MET3p, which is repressed by methionine and cysteine. RESULTS: The reverse-phase HPLC analysis of sterol, extracted from the conditional mutant strain, showed a total lack of ergosterol and instead accumulation of squalene. This imbalance in sterol composition led to defects in growth and increased susceptibilities to drugs including fluconazole, ketoconazole, cycloheximide, nystatin, amphotericin B and terbinafine. Reduced drug efflux activity of the erg1 mutant was associated with poor surface localization of Cdr1p, suggesting that enhanced passive diffusion and reduced efflux mediated by the ABC (ATP binding cassette) transporter Cdr1p increases drug susceptibility. Additionally, conditional erg1 mutant strains were unable to form hyphae in various media. CONCLUSIONS: Taken together, our results demonstrate that the absence of ergosterol, which is one of the constituents of membrane microdomains (rafts), has a direct effect on drug susceptibility and morphogenesis of C. albicans.
Asunto(s)
Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Oxigenasas/fisiología , Difusión , Ergosterol/biosíntesis , Proteínas Fúngicas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Morfogénesis , Mutación , Oxigenasas/genética , Escualeno-MonooxigenasaRESUMEN
Covalent modifications of the amino-termini of the core histones in nucleosomes have been shown to be one of the key regulatory mechanisms in transcription regulation. Recently, new roles for histone modifications have been uncovered for the efficient functioning of RNA Pol II. Besides acetylation, which is the most characterized to date these modifications comprise phosphorylation, methylation, and ubiquitination. This review gives comprehensive view of all the major histone modifications and their effect on transcriptional regulation, in Saccharomyces cerevisiae.
Asunto(s)
Epigénesis Genética , Histonas/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Acetilación , Animales , Histonas/química , Humanos , Metilación , Fosforilación , Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina/metabolismoRESUMEN
Conditions in the infected human host trigger virulence attributes of the fungal pathogen Candida albicans. Specific inducers and elevated temperatures lead to hyphal development or regulate chlamydospore development. To explore if these processes are affected by membrane lipids, an investigation of the functions of the Ole1 fatty acid desaturase (stearoyl-CoA desaturase) in C. albicans, which synthesizes oleic acid, was undertaken. A conditional strain expressing OLE1 from the regulatable MET3 promoter was unable to grow in repressing conditions, indicating that OLE1 is an essential gene. In contrast, a mutant lacking both alleles of OLE2, encoding a Ole1p homologue, was viable and had no apparent phenotypes. Partial repression of MET3p-OLE1 slightly lowered oleic acid levels and decreased membrane fluidity; these conditions permitted growth in the yeast form, but prevented hyphal development in aerobic conditions and blocked the formation of chlamydospores. In contrast, in hypoxic conditions, which trigger an alternative morphogenetic pathway, hyphal morphogenesis was unaffected. Because aerobic morphogenetic signalling and oleic acid biosynthesis require oxygen, it is proposed that oleic acid may function as a sensor activating specific morphogenetic pathways in normoxic conditions.
Asunto(s)
Candida albicans/enzimología , Candida albicans/crecimiento & desarrollo , Regulación Fúngica de la Expresión Génica , Estearoil-CoA Desaturasa/metabolismo , Alelos , Secuencia de Aminoácidos , Candida albicans/genética , Medios de Cultivo , Ácidos Grasos/análisis , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fluidez de la Membrana , Datos de Secuencia Molecular , Morfogénesis , Ácido Oléico/metabolismo , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/genéticaRESUMEN
Fungal APSES proteins regulate morphogenetic processes, including filamentation and differentiation. The human fungal pathogen Candida albicans contains two APSES proteins: the regulator Efg1p and its homologue Efh1p, described here. Overexpression of EFG1 or EFH1 led to similar phenotypes, including pseudohypha formation and opaque-white switching. An efh1 deletion generated no phenotype under most conditions but caused hyperfilamentation in an efg1 background under embedded or hypoxic conditions. This suggests cooperation of these APSES proteins in the suppression of an alternative morphogenetic signaling pathway. Genome-wide transcriptional profiling revealed that EFG1 and EFH1 regulate partially overlapping sets of genes associated with filament formation. Unexpectedly, Efg1p not only regulates genes involved in morphogenesis but also strongly influences the expression of metabolic genes, inducing glycolytic genes and repressing genes essential for oxidative metabolism. Using one- and two-hybrid assays, we further demonstrate that Efg1p is a repressor, whereas Efh1p is an activator of gene expression. Overall, the results suggest that Efh1p supports the regulatory functions of the primary regulator, Efg1p, and indicate a dual role for these APSES proteins in the regulation of fungal morphogenesis and metabolism.
Asunto(s)
Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Candida albicans/genética , Ciclo del Ácido Cítrico/genética , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Glucólisis/genética , Hifa/citología , Datos de Secuencia Molecular , Morfogénesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Estructura Terciaria de Proteína , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Phosphorylation of serine-2 (S2) and serine-5 (S5) of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates the transcription cycle and coordinates recruitment of RNA processing factors. The Fcp1 CTD phosphatase catalyzes dephosphorylation of S2-P. Here, we report that Ssu72, a component of the yeast cleavage/polyadenylation factor (CPF) complex, is a CTD phosphatase with specificity for S5-P. Ssu72 catalyzes CTD S5-P dephosphorylation in association with the Pta1 component of the CPF complex, although its essential role in 3' end processing is independent of catalytic activity. Depletion of Ssu72 impairs transcription in vitro, and this defect can be rescued by recombinant, catalytically active Ssu72. We propose that Ssu72 has a dual role in transcription, one as a CTD S5-P phosphatase that regenerates the initiation-competent, hypophosphorylated form of RNAP II and the other as a factor necessary for cleavage of pre-mRNA and efficient transcription termination.
Asunto(s)
Proteínas Portadoras/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transcripción Genética/genética , Genes Reguladores/genética , Fosfoproteínas Fosfatasas/metabolismo , Estructura Terciaria de Proteína/fisiología , ARN Mensajero/biosíntesis , Saccharomyces cerevisiae/genética , Serina/metabolismo , Especificidad por Sustrato , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
CDR1 gene encoding an ATP-driven drug extrusion pump has been implicated in the development of azole-resistance in Candida albicans. Although the upregulation of CDR1 expression by various environmental factors has been documented, the molecular mechanism underlying such process is poorly understood. We have demonstrated earlier that the CDR1 promoter encompasses a large number of cis-regulatory elements, presumably mediating its response to various drugs. In this study we have identified a novel steroid responsive region (SRR) conferring beta-oestradiol and progesterone inducibility on the CDR1 promoter. The SRR is located -696 to -521 bp upstream of the transcription start site; it is modular in nature and can confer steroid responsiveness to a heterologous promoter (ADH1) linked to a GFP reporter gene. In vitro DNase I protection analyses of SRR revealed two progesterone responsive sequences (-628 to -594 and -683 to -648) and one beta-oestradiol responsive sequence (-628 to -577), which was further corroborated by the gel mobility shift assay. Deletion analyses within the SRR further delimited these steroid responsive sequences into two distinct elements, viz. SRE1 and SRE2. While SRE1 (-677 to -648) responds only to progesterone, SRE2 (-628 to -598) responded to both progesterone and beta-oestradiol. Both SRE1 and SRE2 were specific for steroids, as they did not respond to other drugs, such as cycloheximide, miconazole and terbinafine. In silico comparison of the SRE1/2 with the promoter sequences of other MDR (CDR2 and PDR5) and non-MDR (HSP90) steroid-responsive genes revealed a similarity with respect to conservation of three 5 bp stretches (AAGAA, CCGAA and ATTGG). Taken together, we have identified a novel steroid responsive cis-regulatory sequence in the CDR1 promoter, which presumably can be instrumental in understanding the steroid response cascade in Candida albicans.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Estradiol/farmacología , Proteínas Fúngicas/genética , Proteínas de Transporte de Membrana/genética , Progesterona/farmacología , Secuencia de Bases , Northern Blotting , Candida albicans/metabolismo , Secuencia Conservada , Cicloheximida/farmacología , Huella de ADN , Farmacorresistencia Fúngica/genética , Ensayo de Cambio de Movilidad Electroforética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , Miconazol/farmacología , Datos de Secuencia Molecular , Mutagénesis Insercional , Naftalenos/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Unión Proteica , ARN de Hongos/química , ARN de Hongos/genética , Proteínas Recombinantes , Análisis de Secuencia de ADN , TerbinafinaRESUMEN
The Efg1p regulator protein permits hyphal morphogenesis in the human fungal pathogen Candida albicans. We have identified the major promoter of the EFG1 gene as a direct target of Efg1p, resulting in negative autoregulation of EFG1. Enhanced activity of protein kinase A (PKA) isoforms Tpk1p and Tpk2p or exogenous overexpression of EFG1 led to Efg1p-dependent down-regulation of the endogenous EFG1 promoter. Serial deletion analyses of the promoter region revealed that the TATA box region was required for EFG1 autoregulation. By chromatin immunoprecipitation we detected binding of Efg1p to the EFG1 transcriptional initiation region. Furthermore, Sin3p, a component of a specific histone deacetylase complex, was shown to bind to the EFG1 promoter. sin3 mutants grew as budding pseudohyphae and were unable to form true hyphae, similar to strains constitutively expressing EFG1. We propose that the PKA signalling pathway, in addition to its importance in the initial steps of filament formation, is part of a feedback loop that controls EFG1 expression allowing continued hypha formation in inducing conditions. This autoregulation of EFG1 expression is probably mediated through the Sin3p-containing histone deacetylation complex.