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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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(A) Assembly of a viralgenome maturation and packaging complexat the cos site of the lambda genome. The terminaseprotomer is composed of one large gpA subunit tightly associated withtwo smaller gpNu1 subunits. Four protomers and an indeterminate numberof IHF dimers cooperatively assemble at a cos sequenceof a genome concatemer to engender the packaging motor complex; cos (red dots) represents the junction of two genomes ina concatemer and serves as the packaging initiation site. Terminaseand IHF are depicted as blue and purple circles, respectively, forthe sake of simplicity. The assembled motor nicks the duplex at cosN to yield the 12-base “sticky” end ofthe genome (complex I). This intermediate binds a procapsid to yieldthe functional packaging motor (complex II), which translocates viralDNA into the shell. (B) Detail of the cos regionof the lambda genome. The sequence is multipartite consisting of cosN (nicking) and cosB (binding) subsites; cosB extends from I2 to R1 elements. The gpNu1 subunit specifically interacts with the threeR elements, and several putative IHF consensus sequences have beenidentified (I0–I4). The model duplexes used in this study areindicated in the Figure: cos274 (274 bp), [R3-I1-R2](75 bp), [I2-R3-I1] (75 bp), I1 (27 bp), and R3 (27 bp). (C) Structuralmodels for IHF–DNA nucleoprotein complexes. The left panelshows the crystal structure of IHF bound in a specific complex withthe H′ element of attP (PDB entry 1OWF) showing a duplexbend angle of >160°. The DNA binding site size in this complexis ∼34 bp. The middle panel shows the cocrystal structure of Anabaena HU protein bound in a nonspecific complex (PDBentry 1P71)depicting a “weak” (∼105°) bend in the duplexthat is found in condensed, nucleoid DNA. The DNA binding site sizein this complex is ∼20 bp. The right panel shows the structuralmodel for IHF bound in a nonspecific, linear complex. The model wasconstructed using MacPymol by manually docking the crystal structureof IHF onto the minimal nonspecific R3 duplex. The DNA binding sitesize in this complex is ∼8 bp. In all structures, DNA is coloredcyan and the α and β subunits of IHF are colored lightand dark purple, respectively.
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fig1: (A) Assembly of a viralgenome maturation and packaging complexat the cos site of the lambda genome. The terminaseprotomer is composed of one large gpA subunit tightly associated withtwo smaller gpNu1 subunits. Four protomers and an indeterminate numberof IHF dimers cooperatively assemble at a cos sequenceof a genome concatemer to engender the packaging motor complex; cos (red dots) represents the junction of two genomes ina concatemer and serves as the packaging initiation site. Terminaseand IHF are depicted as blue and purple circles, respectively, forthe sake of simplicity. The assembled motor nicks the duplex at cosN to yield the 12-base “sticky” end ofthe genome (complex I). This intermediate binds a procapsid to yieldthe functional packaging motor (complex II), which translocates viralDNA into the shell. (B) Detail of the cos regionof the lambda genome. The sequence is multipartite consisting of cosN (nicking) and cosB (binding) subsites; cosB extends from I2 to R1 elements. The gpNu1 subunit specifically interacts with the threeR elements, and several putative IHF consensus sequences have beenidentified (I0–I4). The model duplexes used in this study areindicated in the Figure: cos274 (274 bp), [R3-I1-R2](75 bp), [I2-R3-I1] (75 bp), I1 (27 bp), and R3 (27 bp). (C) Structuralmodels for IHF–DNA nucleoprotein complexes. The left panelshows the crystal structure of IHF bound in a specific complex withthe H′ element of attP (PDB entry 1OWF) showing a duplexbend angle of >160°. The DNA binding site size in this complexis ∼34 bp. The middle panel shows the cocrystal structure of Anabaena HU protein bound in a nonspecific complex (PDBentry 1P71)depicting a “weak” (∼105°) bend in the duplexthat is found in condensed, nucleoid DNA. The DNA binding site sizein this complex is ∼20 bp. The right panel shows the structuralmodel for IHF bound in a nonspecific, linear complex. The model wasconstructed using MacPymol by manually docking the crystal structureof IHF onto the minimal nonspecific R3 duplex. The DNA binding sitesize in this complex is ∼8 bp. In all structures, DNA is coloredcyan and the α and β subunits of IHF are colored lightand dark purple, respectively.

Mentions: Terminase enzymes are responsible for viral genome packaging,19 and we have demonstrated that IHF promotes theassembly of a terminase motor complex at cos, thepackaging initiation site in a lambda genome (Figure 1A).18,20 Our lab is interested in thethermodynamic features of packaging motor assembly; unfortunately,this represents a cooperative, multipartite interaction of four heterotrimericterminase protomers and an indeterminate number of IHF proteins withmultiple, putative recognition elements dispersed within the ∼270bp cos sequence (Figure 1B).15,18,21,22 This presents an extremely complex system from which to dissectdetailed mechanistic information. Therefore, as a first step towardbiochemical characterization of these viral genome packaging complexes,we here characterize the most fundamental of these interactions, bindingof IHF to the lambda cos sequence. The results provideinsight into the general mechanism by which IHF can promote the assemblyof specific regulatory complexes in the context of a vast excess ofnonspecific nucleoid formation within the cell.


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)

(A) Assembly of a viralgenome maturation and packaging complexat the cos site of the lambda genome. The terminaseprotomer is composed of one large gpA subunit tightly associated withtwo smaller gpNu1 subunits. Four protomers and an indeterminate numberof IHF dimers cooperatively assemble at a cos sequenceof a genome concatemer to engender the packaging motor complex; cos (red dots) represents the junction of two genomes ina concatemer and serves as the packaging initiation site. Terminaseand IHF are depicted as blue and purple circles, respectively, forthe sake of simplicity. The assembled motor nicks the duplex at cosN to yield the 12-base “sticky” end ofthe genome (complex I). This intermediate binds a procapsid to yieldthe functional packaging motor (complex II), which translocates viralDNA into the shell. (B) Detail of the cos regionof the lambda genome. The sequence is multipartite consisting of cosN (nicking) and cosB (binding) subsites; cosB extends from I2 to R1 elements. The gpNu1 subunit specifically interacts with the threeR elements, and several putative IHF consensus sequences have beenidentified (I0–I4). The model duplexes used in this study areindicated in the Figure: cos274 (274 bp), [R3-I1-R2](75 bp), [I2-R3-I1] (75 bp), I1 (27 bp), and R3 (27 bp). (C) Structuralmodels for IHF–DNA nucleoprotein complexes. The left panelshows the crystal structure of IHF bound in a specific complex withthe H′ element of attP (PDB entry 1OWF) showing a duplexbend angle of >160°. The DNA binding site size in this complexis ∼34 bp. The middle panel shows the cocrystal structure of Anabaena HU protein bound in a nonspecific complex (PDBentry 1P71)depicting a “weak” (∼105°) bend in the duplexthat is found in condensed, nucleoid DNA. The DNA binding site sizein this complex is ∼20 bp. The right panel shows the structuralmodel for IHF bound in a nonspecific, linear complex. The model wasconstructed using MacPymol by manually docking the crystal structureof IHF onto the minimal nonspecific R3 duplex. The DNA binding sitesize in this complex is ∼8 bp. In all structures, DNA is coloredcyan and the α and β subunits of IHF are colored lightand dark purple, respectively.
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fig1: (A) Assembly of a viralgenome maturation and packaging complexat the cos site of the lambda genome. The terminaseprotomer is composed of one large gpA subunit tightly associated withtwo smaller gpNu1 subunits. Four protomers and an indeterminate numberof IHF dimers cooperatively assemble at a cos sequenceof a genome concatemer to engender the packaging motor complex; cos (red dots) represents the junction of two genomes ina concatemer and serves as the packaging initiation site. Terminaseand IHF are depicted as blue and purple circles, respectively, forthe sake of simplicity. The assembled motor nicks the duplex at cosN to yield the 12-base “sticky” end ofthe genome (complex I). This intermediate binds a procapsid to yieldthe functional packaging motor (complex II), which translocates viralDNA into the shell. (B) Detail of the cos regionof the lambda genome. The sequence is multipartite consisting of cosN (nicking) and cosB (binding) subsites; cosB extends from I2 to R1 elements. The gpNu1 subunit specifically interacts with the threeR elements, and several putative IHF consensus sequences have beenidentified (I0–I4). The model duplexes used in this study areindicated in the Figure: cos274 (274 bp), [R3-I1-R2](75 bp), [I2-R3-I1] (75 bp), I1 (27 bp), and R3 (27 bp). (C) Structuralmodels for IHF–DNA nucleoprotein complexes. The left panelshows the crystal structure of IHF bound in a specific complex withthe H′ element of attP (PDB entry 1OWF) showing a duplexbend angle of >160°. The DNA binding site size in this complexis ∼34 bp. The middle panel shows the cocrystal structure of Anabaena HU protein bound in a nonspecific complex (PDBentry 1P71)depicting a “weak” (∼105°) bend in the duplexthat is found in condensed, nucleoid DNA. The DNA binding site sizein this complex is ∼20 bp. The right panel shows the structuralmodel for IHF bound in a nonspecific, linear complex. The model wasconstructed using MacPymol by manually docking the crystal structureof IHF onto the minimal nonspecific R3 duplex. The DNA binding sitesize in this complex is ∼8 bp. In all structures, DNA is coloredcyan and the α and β subunits of IHF are colored lightand dark purple, respectively.
Mentions: Terminase enzymes are responsible for viral genome packaging,19 and we have demonstrated that IHF promotes theassembly of a terminase motor complex at cos, thepackaging initiation site in a lambda genome (Figure 1A).18,20 Our lab is interested in thethermodynamic features of packaging motor assembly; unfortunately,this represents a cooperative, multipartite interaction of four heterotrimericterminase protomers and an indeterminate number of IHF proteins withmultiple, putative recognition elements dispersed within the ∼270bp cos sequence (Figure 1B).15,18,21,22 This presents an extremely complex system from which to dissectdetailed mechanistic information. Therefore, as a first step towardbiochemical characterization of these viral genome packaging complexes,we here characterize the most fundamental of these interactions, bindingof IHF to the lambda cos sequence. The results provideinsight into the general mechanism by which IHF can promote the assemblyof specific regulatory complexes in the context of a vast excess ofnonspecific nucleoid formation within the cell.

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