RESUMO
The yeast CIT1 (mitochondrial citrate synthase) gene is subject to glucose repression and is further repressed by glucose plus glutamate. Based on deletion analysis of a CIT1-lacZ gene fusion, DNA sequences between -548 and -273 are required for full expression of CIT1. The region of transcription initiation and the putative TATA element are located at -150 to -100 and -195 respectively. A restriction fragment containing DNA sequences between -457 and -211 conferred activation and glucose-glutamate regulation when placed in either orientation upstream of a UAS-less heterologous yeast gene. Deletion of DNA sequences between -291 and -273 specifically eliminated derepression of CIT1, and destroyed one of two closely-spaced, potential binding sites for the HAP2,3,4 transcriptional activator protein. Ten-base-pair block substitutions in the region -367 to -348 reduced glucose-repressed expression. Thus, it appears that distinct DNA sequences upstream of CIT1 activate expression in glucose-repressed and derepressed cells. Possible mechanisms of regulation by glutamate plus glucose, are discussed.
Assuntos
Fator de Ligação a CCAAT , Citrato (si)-Sintase/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Ácido Glutâmico/farmacologia , Isoenzimas/genética , Sequências Reguladoras de Ácido Nucleico , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Citrato (si)-Sintase/biossíntese , Ciclo do Ácido Cítrico/efeitos dos fármacos , Sinergismo Farmacológico , Indução Enzimática/efeitos dos fármacos , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/metabolismo , Isoenzimas/biossíntese , Lactatos/farmacologia , Ácido Láctico , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Deleção de Sequência , Fatores de Transcrição/metabolismoRESUMO
The yeast nuclear gene CIT1 encodes mitochondrial citrate synthase, which catalyses the first and rate-limiting step of the tricarboxylic acid (TCA) cycle. Transcription of CIT1 is subject to glucose repression. Mutations in HAP2, HAP3 or HAP4 block derepression of a CIT1-lacZ gene fusion. The HAP2,3,4 transcriptional activator also activates nuclear genes encoding components of the mitochondrial electron transport chain, and thus it co-ordinates derepression of two major mitochondrial functions. Two DNA sequences resembling the consensus HAP2,3,4-binding site (ACCAATNA) are located at approximately -310 and -290, upstream of the CIT1 coding sequence. Deletion and mutation analysis indicates that the -290 element is critical for activation by HAP2,3,4. Glucose-repressed expression of CIT1 is largely independent of HAP2,3,4, is repressed by glutamate, and requires a DNA sequence between -367 and -348. Evidence is presented for a second HAP2,3,4-independent activation element located just upstream and overlapping the -290 HAP2,3,4 element.