Tn10 transpososome assembly involves a folded intermediate that must be unfolded for target capture and strand transfer

Tn10 transposition, like all transposition reactions examined thus far, inv olves assembly of a stable protein-DNA transpososome, containing a pair of transposon ends, within which all chemical events occur. We report here tha t stable Tn10 pre-cleavage transpososomes occur in two conformations: a fol ded form which contains the DNA-bending factor IHF and an unfolded form whi ch lacks IHF, Functional analysis shows that both forms undergo double stra nd cleavage at the transposon ends but that only the unfolded form is compe tent for target capture (and thus for strand transfer to target DNA), Addit ional studies reveal that formation of any type of stable transpososome, fo lded or unfolded, requires not only IHF but also non-specific transposase-D NA contacts immediately internal to the IHF-binding site, implying the occu rrence of a topologically closed loop at the transposon end, Overall, trans pososome assembly must proceed via a folded intermediate which, however, mu st be unfolded in order for intermolecular transposition to occur, These an d other results support key features of a recently proposed model for trans pososome assembly and morphogenesis.