RESUMO
Previously, we demonstrated that transcription-coupled DNA supercoiling (TCDS) potently activated or inhibited nearby promoters in Escherichia coli even in the presence of all four DNA topoisomerases, suggesting that DNA topoisomerases are not the only factors regulating TCDS. A different mechanism exists to confine this localized DNA supercoiling. Using an in vivo system containing the TCDS-activated leu-500 promoter (Pleu-500 ), we find that the nucleoid-associated Fis protein potently inhibits the TCDS-mediated activation of Pleu-500 . We also find that deletion of the fis gene significantly enhances TCDS-mediated inhibition of transcription of three genes purH, yieP, and yrdA divergently coupled to different rrn operons in the early log phase. These results suggest that Fis protein forms DNA topological barriers upon binding to its recognition sites, blocks TCDS diffusion, and potently inhibits the TCDS-activated Pleu-500 .
Assuntos
DNA Super-Helicoidal/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/metabolismo , Regiões Promotoras Genéticas , DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The E. coli gyrA promoter (PgyrA) is a DNA supercoiling sensitive promoter, stimulated by relaxation of DNA templates, and inhibited by (-) DNA supercoiling in bacteria. However, whether PgyrA can be inhibited by transient and localized transcription-coupled DNA supercoiling (TCDS) has not been fully examined. In this paper, using different DNA templates including the E. coli chromosome, we show that transient and localized TCDS strongly inhibits PgyrA in E. coli. This result can be explained by a twin-supercoiled domain model of transcription in which (+) and (-) supercoiled domains are generated around the transcribing RNA polymerase. We also find that fluoroquinolones, such as ciprofloxacin, can substantially increase the expression of the firefly luciferase under the control of the PgyrA coupled to a divergent IPTG-inducible promoter in the presence of IPTG. This stimulation of PgyrA by fluoroquinolones can be also explained by the twin-supercoiled domain model of transcription. This unique property of TCDS may be configured into a high throughput-screening (HTS) assay to identify antimicrobial compounds targeting bacterial DNA gyrase.
Assuntos
DNA Girase/genética , DNA Bacteriano/genética , DNA Super-Helicoidal/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Gênica , Antibacterianos/farmacologia , Sequência de Bases , Cromossomos Bacterianos/química , Cromossomos Bacterianos/metabolismo , Ciprofloxacina/farmacologia , DNA Girase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Genes Reporter , Isopropiltiogalactosídeo/farmacologia , Luciferases/genética , Luciferases/metabolismo , Modelos Genéticos , Novobiocina/farmacologia , Plasmídeos/química , Plasmídeos/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The inactive prokaryotic leu-500 promoter (Pleu-500) contains a single A-to-G point mutation in the -10 region of the leucine operon promoter, which causes leucine auxotrophy. This promoter can be activated by (-) DNA supercoiling in Escherichia coli topA strains. However, whether this activation arises from global, permanent, or transient, dynamic supercoiling is still not fully understood. In this article, using a newly established in vivo system carrying a pair of divergently coupled promoters, i.e. an IPTG-inducible promoter and Pleu-500 that control the expression of lacZ and luc (the firefly luciferase gene), respectively, we demonstrate that transient, dynamic (-) DNA supercoiling provided by divergent transcription in both wild-type and topA strains can potently activate Pleu-500 We found that this activation depended on the promoter strength and the length of RNA transcripts, which are functional characteristics of transcription-coupled DNA supercoiling (TCDS) precisely predicted by the twin-supercoiled domain model of transcription in which a (+) supercoiled domain is produced ahead of the RNA polymerase and a (-) supercoiled domain behind it. We also demonstrate that TCDS can be generated on topologically open DNA molecules, i.e. linear DNA molecules, in Escherichia coli, suggesting that topological boundaries or barriers are not required for the production of TCDS in vivo This work demonstrates that transient, dynamic TCDS by RNA polymerases is a major chromosome remodeling force in E. coli and greatly influences the nearby, coupled promoters/transcription.
Assuntos
DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutação Puntual , Regiões Promotoras Genéticas , Ativação Transcricional , Montagem e Desmontagem da Cromatina , DNA Bacteriano/química , DNA Circular , DNA Recombinante/química , DNA Recombinante/metabolismo , DNA Super-Helicoidal/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Deleção de Genes , Cinética , Leucina/metabolismo , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Óperon , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Transcrição Gênica , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (-) supercoils within the 401-bp DNA loop of the lac promoter and forms a topological barrier. The stability of LacI-mediated DNA topological barriers is directly proportional to its DNA binding affinity. However, we find that DNA supercoiling modulates the basal expression from the lac operon in E. coli. Our results are consistent with the hypothesis that LacI functions as a topological barrier to constrain free, unconstrained (-) supercoils within the 401-bp DNA loop of the lac promoter. These constrained (-) supercoils enhance LacI's DNA-binding affinity and thereby the repression of the promoter. Thus, LacI binding is superhelically modulated to control the expression of lacZYA in the lac operon under varying growth conditions.
