RESUMO
We report that RapA, an Escherichia coli RNA polymerase (RNAP)-associated homolog of SWI2/SNF2, is capable of dramatic activation of RNA synthesis. The RapA-mediated transcriptional activation in vitro depends on supercoiled DNA and high salt concentrations, a condition that is likely to render the DNA superhelix tightly compacted. Moreover, RapA activates transcription by stimulating RNAP recycling. Mutational analyses indicate that the ATPase activity of RapA is essential for its function as a transcriptional activator, and a rapA null mutant exhibits a growth defect on nutrient plates containing high salt concentrations in vivo. Thus, RapA acts as a general transcription factor and an integral component of the transcription machinery. The mode of action of RapA in remodeling posttranscription or posttermination complexes is discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/enzimologia , Proteínas Nucleares , Fosfoproteínas Fosfatases/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/isolamento & purificação , Cromatina/química , DNA Helicases , Primers do DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Expressão Gênica , Conformação de Ácido Nucleico , Fosfoproteínas Fosfatases/isolamento & purificação , Plasmídeos , RNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificaçãoRESUMO
The Escherichia coli rapA gene encodes the RNA polymerase (RNAP)-associated protein RapA, which is a bacterial member of the SWI/SNF helicase-like protein family. We have studied the rapA promoter and its regulation in vivo and determined the interaction between RNAP and the promoter in vitro. We have found that the expression of rapA is growth phase dependent, peaking at the early log phase. The growth phase control of rapA is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most E. coli promoters. We also found that the rapA promoter is subject to growth rate regulation in vivo and that it forms intrinsic unstable initiation complexes with RNAP in vitro. Furthermore, we have shown that a GC-rich or discriminator sequence between the -10 and +1 positions of the rapA promoter is responsible for its growth rate control and the instability of its initiation complexes with RNAP.
Assuntos
Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Proteínas de Bactérias/biossíntese , Sequência de Bases , Códon de Iniciação , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The expression of the Comamonas testosteroni gene, encoding 3beta/17beta-hydroxysteroid dehydrogenase enzyme (3beta/17beta-HSD), was analyzed at the transcriptional level. Northern blot analysis detected a 1 kb transcript in bacterial cells grown in minimum media supplemented with Casamino acids and testosterone. Also this transcript was observed when cells were grown in presence of 1-dehydrotestosterone, androstenedione and 1,4-androstadien-3, 17dione, but not in presence of acetate, citrate, cholic acid, cholesterol, and cortisol. In addition, this effect was dependent on the presence of another carbon source in the growth medium used, revealing catabolite repression.
Assuntos
17-Hidroxiesteroide Desidrogenases/genética , Androgênios/farmacologia , Comamonas testosteroni/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Northern Blotting , Comamonas testosteroni/enzimologia , Meios de Cultura , Meia-Vida , Cinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Comamonas testosteroni can grow on a variety of steroid compounds as the sole carbon and energy source. In a previous study, we cloned and sequenced the testosterone-inducible betahsd gene from C. testosteroni (Genti-Raimondi, S., Tolmasky, M., Patrito, L., Flury, A. and Actis, L., Molecular cloning and expression of the beta-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni. Gene, 1991, 105, 43-49.). Herein we report the cloning and characterization of another steroid-inducible gene (stdC), located 2400 bp upstream of betahsd. Nucleotide sequencing of a region encompassing the stdC gene revealed an open reading frame 546 bp long including the stop codon TGA with significant similarity to the orf4, orf1 and orf4 of unknown function described in the polyhydroxyalkanoic acid (PHA) cluster of Chromatium vinosum, Rhizobium meliloti and Thiocystis violacea, respectively. The aminoacid sequence deduced from the nucleotide sequence predicts a putative protein of 181 amino acids with a molecular weight of 20715 Da. Northern blot experiments indicate that the stdC gene was transcribed as a monocistronic mRNA with an apparent molecular size of 670 nt. The stdC transcript was abundant in C. testosteroni cells grown with different steroid carbon sources harvested in the exponential phase and was found to be under catabolite repression.
