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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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Electrophoretic mobility shift (EMS) studies of bindingof IHFto specific (cos274) and nonspecific (ns274) DNAsubstrates. (A) Representative polyacrylamide gel showing that IHFbinds to the specific cos274 substrate to afforda distinct retarded complex. The positions of free (F) and bound (B)DNA complexes are indicated with arrows at the right of the gel image.The band in the middle of the gel represents a contaminant in theIRDye-labeled duplex (Supporting Information). It is unaffected in the titration study and was not consideredin the calculation of Fbound. (B) Representativepolyacrylamide gel showing that IHF binds to the nonspecific ns274substrate to afford a concentration-dependent shift and smear on thegel. The positions of free (F) DNA and the bound (B) DNA complexesare indicated at the right of the gel image with an arrow and bar,respectively.
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fig2: Electrophoretic mobility shift (EMS) studies of bindingof IHFto specific (cos274) and nonspecific (ns274) DNAsubstrates. (A) Representative polyacrylamide gel showing that IHFbinds to the specific cos274 substrate to afforda distinct retarded complex. The positions of free (F) and bound (B)DNA complexes are indicated with arrows at the right of the gel image.The band in the middle of the gel represents a contaminant in theIRDye-labeled duplex (Supporting Information). It is unaffected in the titration study and was not consideredin the calculation of Fbound. (B) Representativepolyacrylamide gel showing that IHF binds to the nonspecific ns274substrate to afford a concentration-dependent shift and smear on thegel. The positions of free (F) DNA and the bound (B) DNA complexesare indicated at the right of the gel image with an arrow and bar,respectively.

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)

Electrophoretic mobility shift (EMS) studies of bindingof IHFto specific (cos274) and nonspecific (ns274) DNAsubstrates. (A) Representative polyacrylamide gel showing that IHFbinds to the specific cos274 substrate to afforda distinct retarded complex. The positions of free (F) and bound (B)DNA complexes are indicated with arrows at the right of the gel image.The band in the middle of the gel represents a contaminant in theIRDye-labeled duplex (Supporting Information). It is unaffected in the titration study and was not consideredin the calculation of Fbound. (B) Representativepolyacrylamide gel showing that IHF binds to the nonspecific ns274substrate to afford a concentration-dependent shift and smear on thegel. The positions of free (F) DNA and the bound (B) DNA complexesare indicated at the right of the gel image with an arrow and bar,respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Electrophoretic mobility shift (EMS) studies of bindingof IHFto specific (cos274) and nonspecific (ns274) DNAsubstrates. (A) Representative polyacrylamide gel showing that IHFbinds to the specific cos274 substrate to afforda distinct retarded complex. The positions of free (F) and bound (B)DNA complexes are indicated with arrows at the right of the gel image.The band in the middle of the gel represents a contaminant in theIRDye-labeled duplex (Supporting Information). It is unaffected in the titration study and was not consideredin the calculation of Fbound. (B) Representativepolyacrylamide gel showing that IHF binds to the nonspecific ns274substrate to afford a concentration-dependent shift and smear on thegel. The positions of free (F) DNA and the bound (B) DNA complexesare indicated at the right of the gel image with an arrow and bar,respectively.
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