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Tn10 transposition, like all transposition reactions examined thus far, involves assembly of a stable protein-DNA transpososome, containing a pair of transposon ends, within which all chemical events occur. We report here that stable Tn10 pre-cleavage transpososomes occur in two conformations: a folded form which contains the DNA-bending factor IHF and an unfolded form which lacks IHF. Functional analysis shows that both forms undergo double strand cleavage at the transposon ends but that only the unfolded form is competent for target capture (and thus for strand transfer to target DNA). Additional studies reveal that formation of any type of stable transpososome, folded or unfolded, requires not only IHF but also non-specific transposase-DNA contacts immediately internal to the IHF-binding site, implying the occurrence of a topo- logically closed loop at the transposon end. Overall, transpososome assembly must proceed via a folded intermediate which, however, must be unfolded in order for intermolecular transposition to occur. These and other results support key features of a recently proposed model for transpososome assembly and morphogenesis.

Original publication

DOI

10.1093/emboj/19.4.776

Type

Journal article

Journal

Embo j

Publication Date

15/02/2000

Volume

19

Pages

776 - 785

Keywords

Bacterial Proteins, Base Sequence, Binding Sites, DNA Footprinting, DNA Transposable Elements, DNA, Bacterial, DNA-Binding Proteins, Deoxyribonuclease I, Escherichia coli, Integration Host Factors, Models, Biological, Nucleic Acid Conformation, Protein Binding, Protein Conformation