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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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Interrogation of bindingof IHF to minimal substrates using sedimentationvelocity analytical ultracentrifugation (SV-AUC). Increasing concentrationsof IHF were added to the minimal I1 (specific) and R3 (nonspecific)duplex substrates, and their sedimentation behavior was monitoredby SV-AUC as described in Experimental Procedures. The c(s) distribution for eachbinding experiment was calculated using Sedfit. (A) Normalized c(s) profiles for the specific I1 duplex (27 bp). (B) Normalized c(s) profiles for the nonspecific R3 duplex (27 bp). (C)Weight-average sedimentation coefficients for each of the c(s) distributions shown in panels A (redtriangles, I1) and B (black triangles, R3) were calculated using Sedfit and are plotted as a function of IHFconcentration. The red dotted line represents the best fit of theI1 binding data to the nonspecific finite lattice DNA binding model,which does not adequately describe the data. The solid lines representthe best fit of simultaneous (global) analysis of the R3 (black) and I1 (red) binding data to (i) nonspecificfinite lattice DNA binding and (ii) competitive specific/nonspecificfinite lattice DNA models, respectively. The binding parameters derivedfrom global analysis are presented in Table 1.
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fig5: Interrogation of bindingof IHF to minimal substrates using sedimentationvelocity analytical ultracentrifugation (SV-AUC). Increasing concentrationsof IHF were added to the minimal I1 (specific) and R3 (nonspecific)duplex substrates, and their sedimentation behavior was monitoredby SV-AUC as described in Experimental Procedures. The c(s) distribution for eachbinding experiment was calculated using Sedfit. (A) Normalized c(s) profiles for the specific I1 duplex (27 bp). (B) Normalized c(s) profiles for the nonspecific R3 duplex (27 bp). (C)Weight-average sedimentation coefficients for each of the c(s) distributions shown in panels A (redtriangles, I1) and B (black triangles, R3) were calculated using Sedfit and are plotted as a function of IHFconcentration. The red dotted line represents the best fit of theI1 binding data to the nonspecific finite lattice DNA binding model,which does not adequately describe the data. The solid lines representthe best fit of simultaneous (global) analysis of the R3 (black) and I1 (red) binding data to (i) nonspecificfinite lattice DNA binding and (ii) competitive specific/nonspecificfinite lattice DNA models, respectively. The binding parameters derivedfrom global analysis are presented in Table 1.

Mentions: Analysis of the experimental data usingthe models presented aboverequires fitting of multiple parameters that if allowed to float inan unconstrained NLLS analysis would likely contribute to lower precisionin the derived values. To constrain the analysis and provide betterresolved values, the ensemble of AUC data for the R3 and I1 duplexeswas analyzed globally, as follows. The R3 data were modeled to thefinite nonspecific binding model (eq 3a), andthe I1 data were simultaneously fit to the competition binding model(eq 3b). The nonspecific site size (n = 8), duplex length (N = 27), and experimentallydetermined sedimentation coefficient for free DNA (sfree* = 1.96)were held fixed as global constants; ⟨sbound-NS*⟩and sbound-SP* were local variables used in the finite nonspecificbinding and competition binding equations, respectively, and υns, Kns, Ksp, and ω were global variables that were allowed tofloat to their best values by NLLS analytical methods using Scientist(Micromath Scientific Software). The best fits of the data are shownas solid lines in Figure 5C.


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)

Interrogation of bindingof IHF to minimal substrates using sedimentationvelocity analytical ultracentrifugation (SV-AUC). Increasing concentrationsof IHF were added to the minimal I1 (specific) and R3 (nonspecific)duplex substrates, and their sedimentation behavior was monitoredby SV-AUC as described in Experimental Procedures. The c(s) distribution for eachbinding experiment was calculated using Sedfit. (A) Normalized c(s) profiles for the specific I1 duplex (27 bp). (B) Normalized c(s) profiles for the nonspecific R3 duplex (27 bp). (C)Weight-average sedimentation coefficients for each of the c(s) distributions shown in panels A (redtriangles, I1) and B (black triangles, R3) were calculated using Sedfit and are plotted as a function of IHFconcentration. The red dotted line represents the best fit of theI1 binding data to the nonspecific finite lattice DNA binding model,which does not adequately describe the data. The solid lines representthe best fit of simultaneous (global) analysis of the R3 (black) and I1 (red) binding data to (i) nonspecificfinite lattice DNA binding and (ii) competitive specific/nonspecificfinite lattice DNA models, respectively. The binding parameters derivedfrom global analysis are presented in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263431&req=5

fig5: Interrogation of bindingof IHF to minimal substrates using sedimentationvelocity analytical ultracentrifugation (SV-AUC). Increasing concentrationsof IHF were added to the minimal I1 (specific) and R3 (nonspecific)duplex substrates, and their sedimentation behavior was monitoredby SV-AUC as described in Experimental Procedures. The c(s) distribution for eachbinding experiment was calculated using Sedfit. (A) Normalized c(s) profiles for the specific I1 duplex (27 bp). (B) Normalized c(s) profiles for the nonspecific R3 duplex (27 bp). (C)Weight-average sedimentation coefficients for each of the c(s) distributions shown in panels A (redtriangles, I1) and B (black triangles, R3) were calculated using Sedfit and are plotted as a function of IHFconcentration. The red dotted line represents the best fit of theI1 binding data to the nonspecific finite lattice DNA binding model,which does not adequately describe the data. The solid lines representthe best fit of simultaneous (global) analysis of the R3 (black) and I1 (red) binding data to (i) nonspecificfinite lattice DNA binding and (ii) competitive specific/nonspecificfinite lattice DNA models, respectively. The binding parameters derivedfrom global analysis are presented in Table 1.
Mentions: Analysis of the experimental data usingthe models presented aboverequires fitting of multiple parameters that if allowed to float inan unconstrained NLLS analysis would likely contribute to lower precisionin the derived values. To constrain the analysis and provide betterresolved values, the ensemble of AUC data for the R3 and I1 duplexeswas analyzed globally, as follows. The R3 data were modeled to thefinite nonspecific binding model (eq 3a), andthe I1 data were simultaneously fit to the competition binding model(eq 3b). The nonspecific site size (n = 8), duplex length (N = 27), and experimentallydetermined sedimentation coefficient for free DNA (sfree* = 1.96)were held fixed as global constants; ⟨sbound-NS*⟩and sbound-SP* were local variables used in the finite nonspecificbinding and competition binding equations, respectively, and υns, Kns, Ksp, and ω were global variables that were allowed tofloat to their best values by NLLS analytical methods using Scientist(Micromath Scientific Software). The best fits of the data are shownas solid lines in Figure 5C.

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