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Effects of nucleoid proteins on DNA repression loop formation in Escherichia coli.

Becker NA, Kahn JD, Maher LJ - Nucleic Acids Res. (2007)

Bottom Line: Deletion of IHF has little effect.Chloroquine titration, psoralen crosslinking and supercoiling-sensitive reporter assays show that the effects of nucleoid proteins on looping are not correlated with their effects on either total or unrestrained supercoiling.These results suggest that host nucleoid proteins can directly facilitate or inhibit DNA looping in bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First St SW, Rochester, MN 55905, USA.

ABSTRACT
The intrinsic stiffness of DNA limits its ability to be bent and twisted over short lengths, but such deformations are required for gene regulation. One classic paradigm is DNA looping in the regulation of the Escherichia coli lac operon. Lac repressor protein binds simultaneously to two operator sequences flanking the lac promoter. Analysis of the length dependence of looping-dependent repression of the lac operon provides insight into DNA deformation energetics within cells. The apparent flexibility of DNA is greater in vivo than in vitro, possibly because of host proteins that bind DNA and induce sites of flexure. Here we test DNA looping in bacterial strains lacking the nucleoid proteins HU, IHF or H-NS. We confirm that deletion of HU inhibits looping and that quantitative modeling suggests residual looping in the induced operon. Deletion of IHF has little effect. Remarkably, DNA looping is strongly enhanced in the absence of H-NS, and an explanatory model is proposed. Chloroquine titration, psoralen crosslinking and supercoiling-sensitive reporter assays show that the effects of nucleoid proteins on looping are not correlated with their effects on either total or unrestrained supercoiling. These results suggest that host nucleoid proteins can directly facilitate or inhibit DNA looping in bacteria.

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Related in: MedlinePlus

Schematic elements of promoter/reporter constructs for monitoring unrestrained supercoiling in E. coli. Constructs contain firefly luciferase reporters. (A) Luciferase expression is driven from the cluster of topA promoters, induced by high levels of negative superhelical strain and repressed by low levels of negative superhelical strain. (B) Luciferase expression is driven by the gyrA promoter, induced by low levels of negative supercoiling and repressed by high levels of negative supercoiling.
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Figure 4: Schematic elements of promoter/reporter constructs for monitoring unrestrained supercoiling in E. coli. Constructs contain firefly luciferase reporters. (A) Luciferase expression is driven from the cluster of topA promoters, induced by high levels of negative superhelical strain and repressed by low levels of negative superhelical strain. (B) Luciferase expression is driven by the gyrA promoter, induced by low levels of negative supercoiling and repressed by high levels of negative supercoiling.

Mentions: Plasmids pJ1345 (original name pPHB94) and pJ1346 (original name pPHB95) are derived from previously described constructs (61) and were the generous gifts of P. Heisig. Plasmid pJ1345 contains the luciferase gene under control of the topA promoter cluster, while plasmid pJ1346 places the same reporter under the control of the gyrA promoter (Figure 4). For comparisons of unrestrained supercoiling strain in different genetic backgrounds, the topA promoter cluster and gyrA promoter were assayed in two additional contexts. Plasmids pJ1454 and pJ1456 place the promoters on pJ992, a smaller pACYC184-based plasmid for comparison (49,54). Primers 5′-CGAC2G2ATC2T2ATC2GTACTC2TGATG and 5′-GCTCGCTGCAGCG2TGAGA2TG2CA4G) were used to amplify the promoter and reporter regions from plasmid pJ1345 (∼2300 bp product) and pJ1346 (∼2000 bp product), installing flanking BamHI and PstI restriction sites. PCR products were cloned between the BamHI and PstI restriction sites of pJ992 to create plasmid pJ1454 (luciferase gene under the control of the topA promoter) and plasmid pJ1456 (luciferase gene under the control of the gyrA promoter). The promoter-reporters from pJ1454 and pJ1456 were also moved onto the single copy F128 episome by homologous recombination (54). Luciferase assays were performed using the Promega Luciferase assay kit with modifications to accommodate bacterial cells. Briefly, subcultures (5 ml) in LB media containing kanamycin were inoculated with saturated overnight culture (125 µl). Subcultures were grown at 37°C, with agitation, until A600 reached ∼0.6. A sample of the culture (90 µl) was combined with buffer (10 µl: 1 M K2HPO4 pH 7.8, 20 mM EDTA). Samples were mixed and frozen at −80°C for 30 min. To each thawed sample, cell culture lysis reagent (200 µl; Promega, Madison, WI) and fresh lysozyme mix (100 µl of solution containing 5 mg/ml lysozyme and 5 mg/ml BSA) was added. Samples were incubated at 25°C for 10 min. Accurately measured samples (1–5 µl) were added to luminometer tubes followed by the addition of luciferase assay reagent (100 µl; Promega) and luciferase activity was measured on a Turner 20/20 luminometer. The normalized unrestrained supercoiling ratio (Qsc) is given by the topA: gyrA reporter expression ratio, after normalization for cell density:4where A′ is the number of light units per OD600 of culture, and subscripts topA and gyrA refer to the promoters of the luc reporter genes.


Effects of nucleoid proteins on DNA repression loop formation in Escherichia coli.

Becker NA, Kahn JD, Maher LJ - Nucleic Acids Res. (2007)

Schematic elements of promoter/reporter constructs for monitoring unrestrained supercoiling in E. coli. Constructs contain firefly luciferase reporters. (A) Luciferase expression is driven from the cluster of topA promoters, induced by high levels of negative superhelical strain and repressed by low levels of negative superhelical strain. (B) Luciferase expression is driven by the gyrA promoter, induced by low levels of negative supercoiling and repressed by high levels of negative supercoiling.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919473&req=5

Figure 4: Schematic elements of promoter/reporter constructs for monitoring unrestrained supercoiling in E. coli. Constructs contain firefly luciferase reporters. (A) Luciferase expression is driven from the cluster of topA promoters, induced by high levels of negative superhelical strain and repressed by low levels of negative superhelical strain. (B) Luciferase expression is driven by the gyrA promoter, induced by low levels of negative supercoiling and repressed by high levels of negative supercoiling.
Mentions: Plasmids pJ1345 (original name pPHB94) and pJ1346 (original name pPHB95) are derived from previously described constructs (61) and were the generous gifts of P. Heisig. Plasmid pJ1345 contains the luciferase gene under control of the topA promoter cluster, while plasmid pJ1346 places the same reporter under the control of the gyrA promoter (Figure 4). For comparisons of unrestrained supercoiling strain in different genetic backgrounds, the topA promoter cluster and gyrA promoter were assayed in two additional contexts. Plasmids pJ1454 and pJ1456 place the promoters on pJ992, a smaller pACYC184-based plasmid for comparison (49,54). Primers 5′-CGAC2G2ATC2T2ATC2GTACTC2TGATG and 5′-GCTCGCTGCAGCG2TGAGA2TG2CA4G) were used to amplify the promoter and reporter regions from plasmid pJ1345 (∼2300 bp product) and pJ1346 (∼2000 bp product), installing flanking BamHI and PstI restriction sites. PCR products were cloned between the BamHI and PstI restriction sites of pJ992 to create plasmid pJ1454 (luciferase gene under the control of the topA promoter) and plasmid pJ1456 (luciferase gene under the control of the gyrA promoter). The promoter-reporters from pJ1454 and pJ1456 were also moved onto the single copy F128 episome by homologous recombination (54). Luciferase assays were performed using the Promega Luciferase assay kit with modifications to accommodate bacterial cells. Briefly, subcultures (5 ml) in LB media containing kanamycin were inoculated with saturated overnight culture (125 µl). Subcultures were grown at 37°C, with agitation, until A600 reached ∼0.6. A sample of the culture (90 µl) was combined with buffer (10 µl: 1 M K2HPO4 pH 7.8, 20 mM EDTA). Samples were mixed and frozen at −80°C for 30 min. To each thawed sample, cell culture lysis reagent (200 µl; Promega, Madison, WI) and fresh lysozyme mix (100 µl of solution containing 5 mg/ml lysozyme and 5 mg/ml BSA) was added. Samples were incubated at 25°C for 10 min. Accurately measured samples (1–5 µl) were added to luminometer tubes followed by the addition of luciferase assay reagent (100 µl; Promega) and luciferase activity was measured on a Turner 20/20 luminometer. The normalized unrestrained supercoiling ratio (Qsc) is given by the topA: gyrA reporter expression ratio, after normalization for cell density:4where A′ is the number of light units per OD600 of culture, and subscripts topA and gyrA refer to the promoters of the luc reporter genes.

Bottom Line: Deletion of IHF has little effect.Chloroquine titration, psoralen crosslinking and supercoiling-sensitive reporter assays show that the effects of nucleoid proteins on looping are not correlated with their effects on either total or unrestrained supercoiling.These results suggest that host nucleoid proteins can directly facilitate or inhibit DNA looping in bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First St SW, Rochester, MN 55905, USA.

ABSTRACT
The intrinsic stiffness of DNA limits its ability to be bent and twisted over short lengths, but such deformations are required for gene regulation. One classic paradigm is DNA looping in the regulation of the Escherichia coli lac operon. Lac repressor protein binds simultaneously to two operator sequences flanking the lac promoter. Analysis of the length dependence of looping-dependent repression of the lac operon provides insight into DNA deformation energetics within cells. The apparent flexibility of DNA is greater in vivo than in vitro, possibly because of host proteins that bind DNA and induce sites of flexure. Here we test DNA looping in bacterial strains lacking the nucleoid proteins HU, IHF or H-NS. We confirm that deletion of HU inhibits looping and that quantitative modeling suggests residual looping in the induced operon. Deletion of IHF has little effect. Remarkably, DNA looping is strongly enhanced in the absence of H-NS, and an explanatory model is proposed. Chloroquine titration, psoralen crosslinking and supercoiling-sensitive reporter assays show that the effects of nucleoid proteins on looping are not correlated with their effects on either total or unrestrained supercoiling. These results suggest that host nucleoid proteins can directly facilitate or inhibit DNA looping in bacteria.

Show MeSH
Related in: MedlinePlus