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Integration host factor assembly at the cohesive end site of the bacteriophage lambda genome: implications for viral DNA packaging and bacterial gene regulation.

Sanyal SJ, Yang TC, Catalano CE - Biochemistry (2014)

Bottom Line: Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates.At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition.The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington , H-172 Health Sciences Building, Box 357610, Seattle, Washington 98195, United States.

ABSTRACT
Integration host factor (IHF) is an Escherichia coli protein involved in (i) condensation of the bacterial nucleoid and (ii) regulation of a variety of cellular functions. In its regulatory role, IHF binds to a specific sequence to introduce a strong bend into the DNA; this provides a duplex architecture conducive to the assembly of site-specific nucleoprotein complexes. Alternatively, the protein can bind in a sequence-independent manner that weakly bends and wraps the duplex to promote nucleoid formation. IHF is also required for the development of several viruses, including bacteriophage lambda, where it promotes site-specific assembly of a genome packaging motor required for lytic development. Multiple IHF consensus sequences have been identified within the packaging initiation site (cos), and we here interrogate IHF-cos binding interactions using complementary electrophoretic mobility shift (EMS) and analytical ultracentrifugation (AUC) approaches. IHF recognizes a single consensus sequence within cos (I1) to afford a strongly bent nucleoprotein complex. In contrast, IHF binds weakly but with positive cooperativity to nonspecific DNA to afford an ensemble of complexes with increasing masses and levels of condensation. Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates. At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition. The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.

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Quantitative analysis of EMS binding data. TheEMS data (representativedata presented in Figures 2 and 3) were converted to fraction bound DNA versus IHF concentrationas described in Experimental Procedures. (A)Ensemble of EMS data for binding of IHF to the cos274 (blue), [R3-I1-R2] (red), and [I2-R3-I1] (green) duplexes. Eachdata point is the average of at least three separate experiments withthe standard deviation indicated with error bars. The ensemble ofdata was simultaneously analyzed according to the nonspecific finitelattice DNA binding model as described in ExperimentalProcedures. The solid lines represent the best fits of thedata, and the derived binding parameters are presented in Table 1. (B) EMS data for binding of IHF to the minimalI1-specific duplex. Each data point is the average of at least threeseparate experiments with the standard deviation indicated with errorbars. The data were analyzed according to the competitive specific/nonspecificfinite lattice DNA binding model as described in Experimental Procedures. The solid line represents the bestfit of the data, and the derived binding parameters are presentedin Table 1.
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fig4: Quantitative analysis of EMS binding data. TheEMS data (representativedata presented in Figures 2 and 3) were converted to fraction bound DNA versus IHF concentrationas described in Experimental Procedures. (A)Ensemble of EMS data for binding of IHF to the cos274 (blue), [R3-I1-R2] (red), and [I2-R3-I1] (green) duplexes. Eachdata point is the average of at least three separate experiments withthe standard deviation indicated with error bars. The ensemble ofdata was simultaneously analyzed according to the nonspecific finitelattice DNA binding model as described in ExperimentalProcedures. The solid lines represent the best fits of thedata, and the derived binding parameters are presented in Table 1. (B) EMS data for binding of IHF to the minimalI1-specific duplex. Each data point is the average of at least threeseparate experiments with the standard deviation indicated with errorbars. The data were analyzed according to the competitive specific/nonspecificfinite lattice DNA binding model as described in Experimental Procedures. The solid line represents the bestfit of the data, and the derived binding parameters are presentedin Table 1.

Mentions: To constrain the analysis and provide well-resolved parameters,an ensemble of EMS data for cos274, [R3-I1-R2], and[I2-R3-I1] model duplexes (in triplicate, representative data shownin Figures 2A, 3C, and 3D, respectively) were globally fit to eq 1d by nonlinear least-squares (NLLS) analytical methodsusing Scientist (Micromath Scientific Software). The duplex length(N) was held as a local constant for each duplex.The IHF binding site size (n = 8)28 and duplex concentration ([DNA]) were held as global constants. Kns, υns, and ω were globalvariables that were allowed to float to their best values. The bestfit of the ensemble of data is shown as solid lines in Figure 4A.


Integration host factor assembly at the cohesive end site of the bacteriophage lambda genome: implications for viral DNA packaging and bacterial gene regulation.

Sanyal SJ, Yang TC, Catalano CE - Biochemistry (2014)

Quantitative analysis of EMS binding data. TheEMS data (representativedata presented in Figures 2 and 3) were converted to fraction bound DNA versus IHF concentrationas described in Experimental Procedures. (A)Ensemble of EMS data for binding of IHF to the cos274 (blue), [R3-I1-R2] (red), and [I2-R3-I1] (green) duplexes. Eachdata point is the average of at least three separate experiments withthe standard deviation indicated with error bars. The ensemble ofdata was simultaneously analyzed according to the nonspecific finitelattice DNA binding model as described in ExperimentalProcedures. The solid lines represent the best fits of thedata, and the derived binding parameters are presented in Table 1. (B) EMS data for binding of IHF to the minimalI1-specific duplex. Each data point is the average of at least threeseparate experiments with the standard deviation indicated with errorbars. The data were analyzed according to the competitive specific/nonspecificfinite lattice DNA binding model as described in Experimental Procedures. The solid line represents the bestfit of the data, and the derived binding parameters are presentedin Table 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263431&req=5

fig4: Quantitative analysis of EMS binding data. TheEMS data (representativedata presented in Figures 2 and 3) were converted to fraction bound DNA versus IHF concentrationas described in Experimental Procedures. (A)Ensemble of EMS data for binding of IHF to the cos274 (blue), [R3-I1-R2] (red), and [I2-R3-I1] (green) duplexes. Eachdata point is the average of at least three separate experiments withthe standard deviation indicated with error bars. The ensemble ofdata was simultaneously analyzed according to the nonspecific finitelattice DNA binding model as described in ExperimentalProcedures. The solid lines represent the best fits of thedata, and the derived binding parameters are presented in Table 1. (B) EMS data for binding of IHF to the minimalI1-specific duplex. Each data point is the average of at least threeseparate experiments with the standard deviation indicated with errorbars. The data were analyzed according to the competitive specific/nonspecificfinite lattice DNA binding model as described in Experimental Procedures. The solid line represents the bestfit of the data, and the derived binding parameters are presentedin Table 1.
Mentions: To constrain the analysis and provide well-resolved parameters,an ensemble of EMS data for cos274, [R3-I1-R2], and[I2-R3-I1] model duplexes (in triplicate, representative data shownin Figures 2A, 3C, and 3D, respectively) were globally fit to eq 1d by nonlinear least-squares (NLLS) analytical methodsusing Scientist (Micromath Scientific Software). The duplex length(N) was held as a local constant for each duplex.The IHF binding site size (n = 8)28 and duplex concentration ([DNA]) were held as global constants. Kns, υns, and ω were globalvariables that were allowed to float to their best values. The bestfit of the ensemble of data is shown as solid lines in Figure 4A.

Bottom Line: Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates.At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition.The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington , H-172 Health Sciences Building, Box 357610, Seattle, Washington 98195, United States.

ABSTRACT
Integration host factor (IHF) is an Escherichia coli protein involved in (i) condensation of the bacterial nucleoid and (ii) regulation of a variety of cellular functions. In its regulatory role, IHF binds to a specific sequence to introduce a strong bend into the DNA; this provides a duplex architecture conducive to the assembly of site-specific nucleoprotein complexes. Alternatively, the protein can bind in a sequence-independent manner that weakly bends and wraps the duplex to promote nucleoid formation. IHF is also required for the development of several viruses, including bacteriophage lambda, where it promotes site-specific assembly of a genome packaging motor required for lytic development. Multiple IHF consensus sequences have been identified within the packaging initiation site (cos), and we here interrogate IHF-cos binding interactions using complementary electrophoretic mobility shift (EMS) and analytical ultracentrifugation (AUC) approaches. IHF recognizes a single consensus sequence within cos (I1) to afford a strongly bent nucleoprotein complex. In contrast, IHF binds weakly but with positive cooperativity to nonspecific DNA to afford an ensemble of complexes with increasing masses and levels of condensation. Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates. At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition. The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.

Show MeSH
Related in: MedlinePlus