Fis protein forms DNA topological barriers to confine transcription-coupled DNA supercoiling in Escherichia coli.

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 .

Inhibition of the gyrA promoter by transcription-coupled DNA supercoiling in Escherichia coli.

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.

Transient and dynamic DNA supercoiling potently stimulates the leu-500 promoter in Escherichia coli.

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.

DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli.

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.

Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis.

Transcription-coupled DNA supercoiling in defined protein systems and in E. coli topA mutant strains.

Transcription by RNA polymerases can stimulate (-) DNA supercoiling both in vitro and in Escherichia coli topA strains. This phenomenon has been successfully explained by a "twin-supercoiled-domain" model of transcription in which (+) supercoils are produced in front of the transcribing RNA polymerase and (-) supercoils behind it. Previously, it has been shown that certain sequence-specific DNA-binding proteins potently stimulate transcription-coupled DNA supercoiling (TCDS) in an in vitro protein system. These results are consistent with a topological barrier model where certain nucleoprotein complexes can form topological barriers that impede the diffusion and merger of independent chromosomal supercoil domains. Indeed, recent biochemical and single-molecule results demonstrated the existence of nucleoprotein-based DNA topological barriers, which are capable of dividing a DNA molecule into different topological domains. Additionally, recent in vivo studies showed that a transcriptional ensemble (including the transcribing RNA polymerase and the RNA transcript) alone is sufficient to cause a change in local DNA superhelicity. This topological change in local chromosome structure should have a great impact on the conformation and function of critical DNA sequence elements, such as promoters and DNA replication origins. In this article, we will also review recent progress by which TCDS is a critical stimulating force to activate transcription initiation from weak promoters, such as the Salmonella typhimurium leu-500 promoter.

Dependence of transcription-coupled DNA supercoiling on promoter strength in Escherichia coli topoisomerase I deficient strains.

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA in vitro and in vivo. This phenomenon has been nicely explained by a "twin-supercoiled-domain" model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In Escherichia coli topA strains, DNA gyrase selectively converts the positively supercoiled domain into negative supercoils to produce hypernegatively supercoiled DNA. In this article, in order to examine whether promoter strength affects transcription-coupled DNA supercoiling (TCDS), we developed a two-plasmid system in which a linear, non-supercoiled plasmid was used to express lac repressor constitutively while a circular plasmid was used to gage TCDS in E. coli cells. Using this two-plasmid system, we found that TCDS in topA strains is dependent on promoter strength. We also demonstrated that transcription-coupled hypernegative supercoiling of plasmid DNA did not need the expression of a membrane-insertion protein for strong promoters; however, it might require co-transcriptional synthesis of a polypeptide. Furthermore, we found that for weak promoters the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. Our results can be explained by the "twin-supercoiled-domain" model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA.

Determining DNA supercoiling enthalpy by isothermal titration calorimetry.

DNA supercoiling plays a critical role in certain essential DNA transactions, such as DNA replication, recombination, and transcription. For this reason, exploring energetics of DNA supercoiling is fundamentally important for understanding its biological functions. In this paper, using a unique property of DNA intercalators, such as ethidium bromide and daunorubicin, which bind to supercoiled, nicked, and relaxed DNA templates with different DNA-binding enthalpies, we determined DNA supercoiling enthalpy of plasmid pXXZ6, a 4.5 kb plasmid to be about 11.5 kcal/mol per linking number change. This determination allowed us to partition the DNA supercoiling free energy into enthalpic and entropic contributions where the unfavorable DNA supercoiling free energy exclusively originated from the large positive supercoiling enthalpy and was compensated by a large, favorable entropy term (TΔS).