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Cyclic changes in the affinity of protein-DNA interactions drive the progression and regulate the outcome of the Tn10 transposition reaction.

Liu D, Crellin P, Chalmers R - Nucleic Acids Res. (2005)

Bottom Line: During transpososome assembly, IHF is bound with high affinity.However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex.The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford South Parks Road, Oxford OX1 3QU, UK.

ABSTRACT
The Tn10 transpososome is a DNA processing machine in which two transposon ends, a transposase dimer and the host protein integration host factor (IHF), are united in an asymmetrical complex. The transitions that occur during one transposition cycle are not limited to chemical cleavage events at the transposon ends, but also involve a reorganization of the protein and DNA components. Here, we demonstrate multiple pathways for Tn10 transposition. We show that one series of events is favored over all others and involves cyclic changes in the affinity of IHF for its binding site. During transpososome assembly, IHF is bound with high affinity. However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex. The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions.

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Assembly of the Tn10 transpososome and the chemical steps of the reaction. (A) Assembly and unfolding of the synaptic complex. Synapsis of two IHF-bound transposon arms by transposase produces an ffbPEC. Treatment with competitor DNA or heparin strips IHF from the β side of the complex to produce sfbPEC. IHF remains bound to the α side of the complex, presumably due to the subterminal transposase contacts with the transposon end. Divalent metal ion unlocks the IHF binding site on the α side of the complex. Dissociation of IHF produces the tPEC. Hatched oval, IHF; open ovals, transposase; and arrowhead, transposon end. In the tPEC, unoccupied IHF binding sites are indicated by a kink in the transposon end. (B) The chemical steps of the transposition reaction are illustrated using the tPEC as the starting point. In the presence of Mg++, the flanking DNA is cleaved to produce a SEB, followed by a DEB. Non-covalent interactions with a target site are followed by the strand transfer step that produces an insertion product. Asterisk, location of the 32P-label on the transposon end; other elements are as described in (A). (C) The Tn10 transpososome was modeled by superimposing the DNA from the IHF co-crystal structure onto the structure of the Tn5 transpososome (28,32,44,45). Superimposition of the IHF-folded DNA was achieved by minimizing the RMS difference in the position of the equivalent atoms in the Tn5 DNA. One transposon end, IHF and flanking DNA have been omitted for clarity. A section of B DNA (gold) has been docked in the target binding groove to illustrate its spatial relationship to the IHF-folded transposon arm. Regions of transposase and IHF mediated hydroxyl radical protection are shown in red and green, respectively. Every tenth nucleotide on the transferred strand is shown in white. The transposon end is seen embedded in the turquoise monomer of transposase. The subterminal transposase contacts are located on the top of the structure illustrated on the left.
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fig1: Assembly of the Tn10 transpososome and the chemical steps of the reaction. (A) Assembly and unfolding of the synaptic complex. Synapsis of two IHF-bound transposon arms by transposase produces an ffbPEC. Treatment with competitor DNA or heparin strips IHF from the β side of the complex to produce sfbPEC. IHF remains bound to the α side of the complex, presumably due to the subterminal transposase contacts with the transposon end. Divalent metal ion unlocks the IHF binding site on the α side of the complex. Dissociation of IHF produces the tPEC. Hatched oval, IHF; open ovals, transposase; and arrowhead, transposon end. In the tPEC, unoccupied IHF binding sites are indicated by a kink in the transposon end. (B) The chemical steps of the transposition reaction are illustrated using the tPEC as the starting point. In the presence of Mg++, the flanking DNA is cleaved to produce a SEB, followed by a DEB. Non-covalent interactions with a target site are followed by the strand transfer step that produces an insertion product. Asterisk, location of the 32P-label on the transposon end; other elements are as described in (A). (C) The Tn10 transpososome was modeled by superimposing the DNA from the IHF co-crystal structure onto the structure of the Tn5 transpososome (28,32,44,45). Superimposition of the IHF-folded DNA was achieved by minimizing the RMS difference in the position of the equivalent atoms in the Tn5 DNA. One transposon end, IHF and flanking DNA have been omitted for clarity. A section of B DNA (gold) has been docked in the target binding groove to illustrate its spatial relationship to the IHF-folded transposon arm. Regions of transposase and IHF mediated hydroxyl radical protection are shown in red and green, respectively. Every tenth nucleotide on the transferred strand is shown in white. The transposon end is seen embedded in the turquoise monomer of transposase. The subterminal transposase contacts are located on the top of the structure illustrated on the left.

Mentions: Tn10 transposition has been reconstituted in vitro using purified transposase and transposon ends encoded on short linear fragments of DNA (30,31). The synapsis of the transposon ends by transposase into a paired end complex (PEC) requires binding of the host protein IHF to the specific binding site next to the inverted repeat at the outside end of the element (Figure 1A). The 180° bend in the DNA imposed by IHF provides a set of ‘subterminal’ transposase contacts, located distal to the IHF binding site. The terminal and subterminal transposase contacts, therefore, define a loop that constrains the behavior of the bound IHF (see below).


Cyclic changes in the affinity of protein-DNA interactions drive the progression and regulate the outcome of the Tn10 transposition reaction.

Liu D, Crellin P, Chalmers R - Nucleic Acids Res. (2005)

Assembly of the Tn10 transpososome and the chemical steps of the reaction. (A) Assembly and unfolding of the synaptic complex. Synapsis of two IHF-bound transposon arms by transposase produces an ffbPEC. Treatment with competitor DNA or heparin strips IHF from the β side of the complex to produce sfbPEC. IHF remains bound to the α side of the complex, presumably due to the subterminal transposase contacts with the transposon end. Divalent metal ion unlocks the IHF binding site on the α side of the complex. Dissociation of IHF produces the tPEC. Hatched oval, IHF; open ovals, transposase; and arrowhead, transposon end. In the tPEC, unoccupied IHF binding sites are indicated by a kink in the transposon end. (B) The chemical steps of the transposition reaction are illustrated using the tPEC as the starting point. In the presence of Mg++, the flanking DNA is cleaved to produce a SEB, followed by a DEB. Non-covalent interactions with a target site are followed by the strand transfer step that produces an insertion product. Asterisk, location of the 32P-label on the transposon end; other elements are as described in (A). (C) The Tn10 transpososome was modeled by superimposing the DNA from the IHF co-crystal structure onto the structure of the Tn5 transpososome (28,32,44,45). Superimposition of the IHF-folded DNA was achieved by minimizing the RMS difference in the position of the equivalent atoms in the Tn5 DNA. One transposon end, IHF and flanking DNA have been omitted for clarity. A section of B DNA (gold) has been docked in the target binding groove to illustrate its spatial relationship to the IHF-folded transposon arm. Regions of transposase and IHF mediated hydroxyl radical protection are shown in red and green, respectively. Every tenth nucleotide on the transferred strand is shown in white. The transposon end is seen embedded in the turquoise monomer of transposase. The subterminal transposase contacts are located on the top of the structure illustrated on the left.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1074725&req=5

fig1: Assembly of the Tn10 transpososome and the chemical steps of the reaction. (A) Assembly and unfolding of the synaptic complex. Synapsis of two IHF-bound transposon arms by transposase produces an ffbPEC. Treatment with competitor DNA or heparin strips IHF from the β side of the complex to produce sfbPEC. IHF remains bound to the α side of the complex, presumably due to the subterminal transposase contacts with the transposon end. Divalent metal ion unlocks the IHF binding site on the α side of the complex. Dissociation of IHF produces the tPEC. Hatched oval, IHF; open ovals, transposase; and arrowhead, transposon end. In the tPEC, unoccupied IHF binding sites are indicated by a kink in the transposon end. (B) The chemical steps of the transposition reaction are illustrated using the tPEC as the starting point. In the presence of Mg++, the flanking DNA is cleaved to produce a SEB, followed by a DEB. Non-covalent interactions with a target site are followed by the strand transfer step that produces an insertion product. Asterisk, location of the 32P-label on the transposon end; other elements are as described in (A). (C) The Tn10 transpososome was modeled by superimposing the DNA from the IHF co-crystal structure onto the structure of the Tn5 transpososome (28,32,44,45). Superimposition of the IHF-folded DNA was achieved by minimizing the RMS difference in the position of the equivalent atoms in the Tn5 DNA. One transposon end, IHF and flanking DNA have been omitted for clarity. A section of B DNA (gold) has been docked in the target binding groove to illustrate its spatial relationship to the IHF-folded transposon arm. Regions of transposase and IHF mediated hydroxyl radical protection are shown in red and green, respectively. Every tenth nucleotide on the transferred strand is shown in white. The transposon end is seen embedded in the turquoise monomer of transposase. The subterminal transposase contacts are located on the top of the structure illustrated on the left.
Mentions: Tn10 transposition has been reconstituted in vitro using purified transposase and transposon ends encoded on short linear fragments of DNA (30,31). The synapsis of the transposon ends by transposase into a paired end complex (PEC) requires binding of the host protein IHF to the specific binding site next to the inverted repeat at the outside end of the element (Figure 1A). The 180° bend in the DNA imposed by IHF provides a set of ‘subterminal’ transposase contacts, located distal to the IHF binding site. The terminal and subterminal transposase contacts, therefore, define a loop that constrains the behavior of the bound IHF (see below).

Bottom Line: During transpososome assembly, IHF is bound with high affinity.However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex.The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford South Parks Road, Oxford OX1 3QU, UK.

ABSTRACT
The Tn10 transpososome is a DNA processing machine in which two transposon ends, a transposase dimer and the host protein integration host factor (IHF), are united in an asymmetrical complex. The transitions that occur during one transposition cycle are not limited to chemical cleavage events at the transposon ends, but also involve a reorganization of the protein and DNA components. Here, we demonstrate multiple pathways for Tn10 transposition. We show that one series of events is favored over all others and involves cyclic changes in the affinity of IHF for its binding site. During transpososome assembly, IHF is bound with high affinity. However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex. The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions.

Show MeSH
Related in: MedlinePlus