RESUMO
Gene CPA1, encoding one of the subunits of carbamoylphosphate synthetase (CPSase A) is subject to a translational control by arginine of which the essential element is a 25 amino acid peptide encoded by the CPA1 messenger. The peptide is the product of an open reading frame located upstream (uORF) of the coding phase of the gene, within a 250 nucleotide leader. In the past, a series of mutations impairing the repression of gene CPA1 by arginine had been selected in vivo. Most of the mutations were located in the CPA1 uORF, but mutations unlinked to the CPA1 gene were also isolated and mapped in a gene called CPAR. In this work, we show that the CPAR gene is identical to the UPF1 gene, encoding a protein responsible for the premature termination step of RNA surveillance by nonsense-mediated mRNA decay (NMD). Deletion of UPF1, or deletion of UPF2 and UPF3, the other genes involved in the NMD pathway, enhances the synthesis of CPSase A, whether arginine is present or not in the growth medium. The regulatory effect of the NMD protein complex is only observed when the uORF is present in the CPA1 messenger, indicating that the arginine-peptide repression mechanism and the RNA surveillance pathway are complementary mechanisms. Our results indicate that the NMD destabilizes the 5' end of the CPA1 message and this decay is strongly enhanced when arginine is present in the growth medium.
Assuntos
Regulação Fúngica da Expressão Gênica , Genes Fúngicos , RNA Helicases/genética , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal , Arginina/fisiologia , Sequência de Bases , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/biossíntese , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Fases de Leitura Aberta , Iniciação Traducional da Cadeia Peptídica , Mutação Puntual , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The products of three genes named CARGRI, CARGRII, and CARGRIII were shown to repress the expression of CAR1 and CAR2 genes, involved in arginine catabolism. CARGRI is identical to UME6 and encodes a regulator of early meiotic genes. In this work we identify CARGRII as SIN3 and CARGRIII as RPD3. The associated gene products are components of a high-molecular-weight complex with histone deacetylase activity and are recruited by Ume6 to promoters containing a URS1 sequence. Sap30, another component of this complex, is also required to repress CAR1 expression. This histone deacetylase complex prevents the synthesis of the two arginine catabolic enzymes, arginase (CAR1) and ornithine transaminase (CAR2), as long as exogenous nitrogen is available. Upon nitrogen depletion, repression at URS1 is released and Ume6 interacts with ArgRI and ArgRII, two proteins involved in arginine-dependent activation of CAR1 and CAR2, leading to high levels of the two catabolic enzymes despite a low cytosolic arginine pool. Our data also show that the deletion of the UME6 gene impairs cell growth more strongly than the deletion of the SIN3 or RPD3 gene, especially in the Sigma1278b background.
Assuntos
Arginase/genética , Arginina/metabolismo , Genes Fúngicos , Ornitina-Oxo-Ácido Transaminase/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Indução Enzimática , Proteínas Fúngicas/genética , Regulação Bacteriana da Expressão Gênica , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Proteínas de Membrana/genética , Proteína 1 de Manutenção de Minicromossomo , Nitrogênio/deficiência , Ligação Proteica , Proteínas Repressoras/metabolismo , Deleção de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We report here the DNA sequence of a segment (alpha 1006.13: YBLO5) of chromosome II of Saccharomyces cerevisiae, extending over 32.5 kb. The segment contains 26 open reading frames (ORFs) from YBLO501 to YBLO526. YBL0505 corresponds to the SEC17 gene and YBL0521 to the KIP1 gene. YBL0516 contains an intron, YBL0513 shows homology with the RAT protein phosphatase and YBL0526 contains a zinc-finger motif. Disruption of 14 genes by insertion of a URA3 cassette has been performed and these mutants were analysed for their mating and sporulation ability, and for their growth on different carbon sources. YBL0515 and YBL0526 ORFs seem to be involved in the sporulation process.