RESUMO
Budding yeast (Saccharomyces cerevisiae) responds to iron deprivation both by Aft1-Aft2-dependent transcriptional activation of genes involved in cellular iron uptake and by Cth1-Cth2-specific degradation of certain mRNAs coding for iron-dependent biosynthetic components. Here, we provide evidence for a novel principle of iron-responsive gene expression. This regulatory mechanism is based on the modulation of transcription through the iron-dependent variation of levels of regulatory metabolites. As an example, the LEU1 gene of branched-chain amino acid biosynthesis is downregulated under iron-limiting conditions through depletion of the metabolic intermediate alpha-isopropylmalate, which functions as a key transcriptional coactivator of the Leu3 transcription factor. Synthesis of alpha-isopropylmalate involves the iron-sulfur protein Ilv3, which is inactivated under iron deficiency. As another example, decreased mRNA levels of the cytochrome c-encoding CYC1 gene under iron-limiting conditions involve heme-dependent transcriptional regulation via the Hap1 transcription factor. Synthesis of the iron-containing heme is directly correlated with iron availability. Thus, the iron-responsive expression of genes that are downregulated under iron-limiting conditions is conferred by two independent regulatory mechanisms: transcriptional regulation through iron-responsive metabolites and posttranscriptional mRNA degradation. Only the combination of the two processes provides a quantitative description of the response to iron deprivation in yeast.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Heme/metabolismo , Homeostase/fisiologia , Ferro/metabolismo , Malatos/metabolismo , Saccharomyces cerevisiae/metabolismo , 3-Isopropilmalato Desidrogenase/genética , Fator de Ligação a CCAAT/genética , Ceruloplasmina/genética , Citocromos c/genética , Citocromos c/metabolismo , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Ferroquelatase/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Hidroliases/genética , Hidroliases/metabolismo , Quelantes de Ferro/farmacologia , Deficiências de Ferro , Isomerases/genética , Isomerases/metabolismo , Malatos/farmacologia , Peroxidases/genética , Fenantrolinas/farmacologia , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiões Terminadoras Genéticas/genética , Transativadores/genética , Fatores de Transcrição/genética , Tristetraprolina/genética , Regulação para Cima/genéticaRESUMO
We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel beta-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs.