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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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In vivo DNA looping assay. (A) Arrangement of strong (Osym) and weak (O2) lac operators (rectangles) flanking the lac promoter (P) upstream of the lacZ reporter gene. Operator spacing was systematically varied from 63 to 91 bp in individual strains. The binding face recognized by repressor tetramer is indicated by shading. (B) The Lac repressor tetramer (circles) fills the high-affinity Osym operator, but repression by looping to the O2 operator depends on the properties of the intervening DNA. Looping is disfavored by the energetic costs of twisting and bending DNA. Spacings that position operators on opposite faces of the DNA helix are unfavorable for repression loops because of the requirement for DNA twisting. (C) Spacings that position operators on the same face of DNA are favorable for repression loop formation.
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Figure 1: In vivo DNA looping assay. (A) Arrangement of strong (Osym) and weak (O2) lac operators (rectangles) flanking the lac promoter (P) upstream of the lacZ reporter gene. Operator spacing was systematically varied from 63 to 91 bp in individual strains. The binding face recognized by repressor tetramer is indicated by shading. (B) The Lac repressor tetramer (circles) fills the high-affinity Osym operator, but repression by looping to the O2 operator depends on the properties of the intervening DNA. Looping is disfavored by the energetic costs of twisting and bending DNA. Spacings that position operators on opposite faces of the DNA helix are unfavorable for repression loops because of the requirement for DNA twisting. (C) Spacings that position operators on the same face of DNA are favorable for repression loop formation.

Mentions: We have developed an experimental system for measuring DNA flexibility in living E. coli cells and have used it to determine whether proteins play important roles in enhancing the apparent flexibility of DNA. The system, shown schematically in Figure 1, is based on classic studies of DNA looping in repression of the E. coli lactose operon (45–48). The reporter construct is a simplified lac operon, with a lacZ reporter gene placed downstream from the moderately strong lac UV5 promoter. The operon is modified so as to increase sensitivity to DNA looping by using a weak proximal O2 operator to mediate repression and a strong distal Osym operator upstream. The Lac repressor binds strongly to Osym, and it can form a repression loop by simultaneously binding at O2. The stability of this loop is related to the energetic costs of bending and twisting the intervening DNA, which are, in turn, dependent on the distance of separation and helical phasing of the operators. The reporter is introduced in single copy on an F′ episome. Host strains with or without the genes encoding architectural DNA-binding proteins are used; they all express wild-type levels of the wild-type bidentate Lac repressor tetramer (49). The development of this system and its application to flexibility induced by the rat HMG-B protein have been described in detail (49).Figure 1.


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

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

In vivo DNA looping assay. (A) Arrangement of strong (Osym) and weak (O2) lac operators (rectangles) flanking the lac promoter (P) upstream of the lacZ reporter gene. Operator spacing was systematically varied from 63 to 91 bp in individual strains. The binding face recognized by repressor tetramer is indicated by shading. (B) The Lac repressor tetramer (circles) fills the high-affinity Osym operator, but repression by looping to the O2 operator depends on the properties of the intervening DNA. Looping is disfavored by the energetic costs of twisting and bending DNA. Spacings that position operators on opposite faces of the DNA helix are unfavorable for repression loops because of the requirement for DNA twisting. (C) Spacings that position operators on the same face of DNA are favorable for repression loop formation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: In vivo DNA looping assay. (A) Arrangement of strong (Osym) and weak (O2) lac operators (rectangles) flanking the lac promoter (P) upstream of the lacZ reporter gene. Operator spacing was systematically varied from 63 to 91 bp in individual strains. The binding face recognized by repressor tetramer is indicated by shading. (B) The Lac repressor tetramer (circles) fills the high-affinity Osym operator, but repression by looping to the O2 operator depends on the properties of the intervening DNA. Looping is disfavored by the energetic costs of twisting and bending DNA. Spacings that position operators on opposite faces of the DNA helix are unfavorable for repression loops because of the requirement for DNA twisting. (C) Spacings that position operators on the same face of DNA are favorable for repression loop formation.
Mentions: We have developed an experimental system for measuring DNA flexibility in living E. coli cells and have used it to determine whether proteins play important roles in enhancing the apparent flexibility of DNA. The system, shown schematically in Figure 1, is based on classic studies of DNA looping in repression of the E. coli lactose operon (45–48). The reporter construct is a simplified lac operon, with a lacZ reporter gene placed downstream from the moderately strong lac UV5 promoter. The operon is modified so as to increase sensitivity to DNA looping by using a weak proximal O2 operator to mediate repression and a strong distal Osym operator upstream. The Lac repressor binds strongly to Osym, and it can form a repression loop by simultaneously binding at O2. The stability of this loop is related to the energetic costs of bending and twisting the intervening DNA, which are, in turn, dependent on the distance of separation and helical phasing of the operators. The reporter is introduced in single copy on an F′ episome. Host strains with or without the genes encoding architectural DNA-binding proteins are used; they all express wild-type levels of the wild-type bidentate Lac repressor tetramer (49). The development of this system and its application to flexibility induced by the rat HMG-B protein have been described in detail (49).Figure 1.

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