RESUMEN
Gene expression profiling is rapidly becoming a mainstay of functional genomic studies. However, there have been relatively few studies of how the data from expression profiles integrate with more classic approaches to examine gene expression. This study used gene expression profiling of a portion of the genome of Saccharomyces cerevisiae to explore the impact of blocks in the isoprenoid biosynthetic pathway on the expression of genes and the regulation of this pathway. Approximately 50% of the genes whose expression was altered by blocks in isoprenoid biosynthesis were genes previously known to participate in the pathway. In contrast to this simple correspondence, the regulatory patterns revealed by different blocks, and in particular by antifungal azoles, was complex in a manner not anticipated by earlier studies.
Asunto(s)
Proteínas Fúngicas , Fosfatos de Poliisoprenilo/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Inhibidores Enzimáticos del Citocromo P-450 , Cartilla de ADN/genética , Inhibidores Enzimáticos/farmacología , Ergosterol/biosíntesis , Ergosterol/genética , Retroalimentación , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Fúngicos , Genes Reporteros , Genoma Fúngico , Factor de Apareamiento , Oxidorreductasas/antagonistas & inhibidores , Péptidos/genética , Feromonas/genética , Fosfatos de Poliisoprenilo/química , Esterol 14-Desmetilasa , Proteínas ras/genéticaRESUMEN
Sterols and all nonsterol isoprenoids are derived from the highly conserved mevalonate pathway. In animal cells, this pathway is regulated in part at the transcriptional level through the action of sterol response element-binding proteins acting at specific DNA sequences near promoters. Here we extend at least part of this regulatory paradigm to the ERG10 gene, which encodes a sterol-biosynthetic enzyme of Saccharomyces cerevisiae. Specifically, the discovery of sterol-mediated feedback control of ERG10 transcription is reported. Deletion analysis of the ERG10 promoter region identified sequences involved in the expression of ERG10. This regulatory axis appeared to involve sterol levels, as a late block in the pathway that depletes sterol, but not nonsterol isoprenoids, was able to elicit the regulatory response.
Asunto(s)
Coenzima A Ligasas/genética , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Esteroles/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Retroalimentación , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Ácido Mevalónico/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , ARN Mensajero/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Hydroxymethylglutaryl-CoA reductase (HMG-R) is a key enzyme in the mevalonate pathway, from which thousands of molecules are derived including cholesterol and prenyl moieties. The regulation of HMG-R is complex and includes feedback control, cross-regulation by independent bio-chemical processes and contra-regulation of separate isozymes. From studies in yeast, these separate modes of regulation can be considered in an integrated fashion.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/fisiología , Isoenzimas/fisiología , Retroalimentación , Hidroximetilglutaril-CoA Reductasas/genética , Familia de Multigenes , Saccharomyces cerevisiae/enzimologíaRESUMEN
The ability of Ras proteins to initiate eukaryotic cell proliferation requires the post-translational attachment of a farnesyl group, an isoprenoid lipid moiety derived from mevalonate, to the carboxyl-terminus of the protein. This modification is essential for the subsequent processing and intracellular targeting of the Ras protein. Here we report that mevalonate is also required for the efficient synthesis of Ras proteins in Saccharomyces cerevisiae. Depletion of intracellular mevalonate resulted in decreased steady-state levels of Ras1p and Ras2p, an effect that was mediated at the level of mRNA accumulation. The sequences controlling the response of RAS2 mRNA level to mevalonate availability, mapped to the coding region of the RAS2 gene. Mevalonate starvation also had a significant effect on the expression of some, but not all, genes encoding prenylated proteins. The regulatory effect on RAS2 mRNA did not require a functional farnesyl transferase. These results uncover a novel regulatory role for mevalonate-derived products and expand the potential for inhibitors of mevalonate metabolism as anti-cancer agents.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Ácido Mevalónico/metabolismo , Procesamiento Proteico-Postraduccional , ARN de Hongos/biosíntesis , ARN Mensajero/biosíntesis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas ras , Secuencia de Bases , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Datos de Secuencia Molecular , Prenilación de Proteína , ARN de Hongos/genética , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.