RESUMO
Transcriptional regulation of the put operon is mediated by a unique mechanism involving autogenous regulation by the PutA protein, a membrane-associated dehydrogenase. The 420-bp put control region contains the putP and putA promoters, multiple operator sites, multiple catabolite repression protein binding sites, and several potential integration host factor (IHF)-binding sites (ihf). In this study, we show that IHF facilitates repression of the put operon in vivo, and IHF binds specifically to two ihf sites in the put control region in vitro. DNA gyrase mutants that alter the degree of chromosomal supercoiling do not affect put regulation, indicating that the effect of IHF on put expression is in this case independent of supercoiling.
Assuntos
Proteínas de Bactérias/genética , Repressão Enzimática/genética , Óperon/genética , Prolina/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Salmonella typhimurium/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Operadoras Genéticas/genética , Regiões Promotoras Genéticas/genética , Homologia de Sequência do Ácido NucleicoRESUMO
The proline utilization (put) operon of Salmonella typhimurium is transcriptionally repressed by PutA protein in the absence of proline. PutA protein also carries out the enzymatic steps in proline catabolism. These two roles require different cellular localizations of PutA. Catabolism of proline requires PutA to associate with the membrane because reoxidation of the FAD cofactor in PutA needs the presence of an electron acceptor. Repression of the put operon requires PutA to bind to the put control-region DNA in the cytoplasm. The presence of proline, the inducer, is necessary but not sufficient for PutA to discriminate between its roles as an enzyme or as a repressor. Two conditions that prevent PutA protein binding to the put control region are (i) when proline and an electron acceptor or the cytoplasmic membrane are present or (ii) when PutA is reduced by dithionite. These two conditions increase the relative hydrophobicity of PutA protein, favoring membrane association and therefore enzymatic activity.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Proteínas de Membrana/metabolismo , Óperon , Prolina Oxidase/metabolismo , Prolina/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Membrana Celular/enzimologia , Proteínas de Ligação a DNA/genética , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Oxirredução , Prolina Oxidase/genética , Prolina Oxidase/isolamento & purificação , Conformação Proteica , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The PutA protein is a membrane-associated enzyme that catalyzes the degradation of proline to glutamate. Genetic evidence suggests that in the absence of proline, the PutA protein also represses transcription of the putA and putP genes. To directly determine whether PutA protein binds to the put control region, we analyzed gel retardation of put control region DNA by purified PutA protein in vitro. The put control region is 420 bp. Purified PutA protein bound specifically to several nonoverlapping fragments of control region DNA, indicating the presence of multiple binding sites in the control region. Electrophoretic abnormalities and behavior of circularly permuted fragments of control region DNA indicate that it contains a region of intrinsically curved DNA. To determine whether the multiple binding sites or the DNA curvature are important in vivo, two types of deletions were constructed: (i) deletions that removed sequences predicted to contribute to DNA curvature as well as potential operator sites and (ii) deletions that removed only potential operator sites. Both types of deletions increased expression of the put genes but were still induced by proline, indicating that multiple cis elements are involved in repression. These data suggest a model for put repression that invokes the formation of a complex between PutA protein molecules bound at different sites in the control region, brought into proximity by a loop of curved DNA.