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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 full-length cos274 and ns274 duplex substratesusing sedimentation velocity analyticalultracentrifugation (SV-AUC). Increasing concentrations of IHF wereadded to each duplex, and their sedimentation behavior was monitoredby SV-AUC as described in Experimental Procedures. The c(s) distribution for eachbinding experiment were calculated using Sedfit. (A) Normalized c(s) profiles for the specific cos274 duplex. (B) Normalized c(s) profiles for the nonspecific ns274 duplex. (C) Weight-averagesedimentation coefficients for each of the c(s) distributions shown in panel A (red circles, cos274) and panel B (black circles, ns274) were calculatedusing Sedfit and are plotted as a function of IHF concentration. Thesolid lines represent the best fits of simultaneous (global) analysisof the ensemble of binding data to the DNA unbending model (Scheme 1) as described in SupportingInformation. The binding parameters derived from global analysisare presented in Table S2 of the Supporting Information.
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fig6: Interrogation of bindingof IHF to full-length cos274 and ns274 duplex substratesusing sedimentation velocity analyticalultracentrifugation (SV-AUC). Increasing concentrations of IHF wereadded to each duplex, and their sedimentation behavior was monitoredby SV-AUC as described in Experimental Procedures. The c(s) distribution for eachbinding experiment were calculated using Sedfit. (A) Normalized c(s) profiles for the specific cos274 duplex. (B) Normalized c(s) profiles for the nonspecific ns274 duplex. (C) Weight-averagesedimentation coefficients for each of the c(s) distributions shown in panel A (red circles, cos274) and panel B (black circles, ns274) were calculatedusing Sedfit and are plotted as a function of IHF concentration. Thesolid lines represent the best fits of simultaneous (global) analysisof the ensemble of binding data to the DNA unbending model (Scheme 1) as described in SupportingInformation. The binding parameters derived from global analysisare presented in Table S2 of the Supporting Information.

Mentions: We next utilized SV-AUC to examinebinding of IHF to the full-length cos274 and ns274model duplexes. The SV-AUC data were analyzed using Sedfit, and the c(s) distributions are shown in Figure 6. Analysis of the data in the absence of IHF yieldsexperimental sedimentation coefficients of 4.1 for both the cos274 and ns274 duplexes. Incremental addition of IHF tothe specific cos274 duplex results in a progressive,concentration-dependent increase in the experimental sedimentationcoefficient to a maximal value of ∼10 S (Figure 6A). Given the magnitude of the sedimentation coefficient andconsidering the high concentration of IHF required to reach saturation,it is likely that this complex reflects not only specific bindingof IHF to the I1 element but also the assembly ofmultiple IHF dimers in a nonspecific manner. As noted above, thesesubsequent binding events are not detected in the EMS studies exceptat IHF concentrations of >1 μM.


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 full-length cos274 and ns274 duplex substratesusing sedimentation velocity analyticalultracentrifugation (SV-AUC). Increasing concentrations of IHF wereadded to each duplex, and their sedimentation behavior was monitoredby SV-AUC as described in Experimental Procedures. The c(s) distribution for eachbinding experiment were calculated using Sedfit. (A) Normalized c(s) profiles for the specific cos274 duplex. (B) Normalized c(s) profiles for the nonspecific ns274 duplex. (C) Weight-averagesedimentation coefficients for each of the c(s) distributions shown in panel A (red circles, cos274) and panel B (black circles, ns274) were calculatedusing Sedfit and are plotted as a function of IHF concentration. Thesolid lines represent the best fits of simultaneous (global) analysisof the ensemble of binding data to the DNA unbending model (Scheme 1) as described in SupportingInformation. The binding parameters derived from global analysisare presented in Table S2 of the Supporting Information.
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Show All Figures
getmorefigures.php?uid=PMC4263431&req=5

fig6: Interrogation of bindingof IHF to full-length cos274 and ns274 duplex substratesusing sedimentation velocity analyticalultracentrifugation (SV-AUC). Increasing concentrations of IHF wereadded to each duplex, and their sedimentation behavior was monitoredby SV-AUC as described in Experimental Procedures. The c(s) distribution for eachbinding experiment were calculated using Sedfit. (A) Normalized c(s) profiles for the specific cos274 duplex. (B) Normalized c(s) profiles for the nonspecific ns274 duplex. (C) Weight-averagesedimentation coefficients for each of the c(s) distributions shown in panel A (red circles, cos274) and panel B (black circles, ns274) were calculatedusing Sedfit and are plotted as a function of IHF concentration. Thesolid lines represent the best fits of simultaneous (global) analysisof the ensemble of binding data to the DNA unbending model (Scheme 1) as described in SupportingInformation. The binding parameters derived from global analysisare presented in Table S2 of the Supporting Information.
Mentions: We next utilized SV-AUC to examinebinding of IHF to the full-length cos274 and ns274model duplexes. The SV-AUC data were analyzed using Sedfit, and the c(s) distributions are shown in Figure 6. Analysis of the data in the absence of IHF yieldsexperimental sedimentation coefficients of 4.1 for both the cos274 and ns274 duplexes. Incremental addition of IHF tothe specific cos274 duplex results in a progressive,concentration-dependent increase in the experimental sedimentationcoefficient to a maximal value of ∼10 S (Figure 6A). Given the magnitude of the sedimentation coefficient andconsidering the high concentration of IHF required to reach saturation,it is likely that this complex reflects not only specific bindingof IHF to the I1 element but also the assembly ofmultiple IHF dimers in a nonspecific manner. As noted above, thesesubsequent binding events are not detected in the EMS studies exceptat IHF concentrations of >1 μM.

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