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

Abstract

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.

The global regulator H-NS acts directly on the transpososome to promote Tn10 transposition

“…5B). This experiment demonstrates that occupancy of IHF sites is low in this transpososome, which is indicative of an unfolded transpososome (Sakai et al 2000;Crellin and Chalmers 2001). With this substrate IHF binding is revealed by hypersensitivity to OP-Cu at nucleotides 27 and 28, and by OP-Cu protection in the region of nucleotides 31-45 on the transferred strand (Fig.…”

“…We have seen no evidence of this in footprinting studies (data not shown). Moreover, since we know that the conformation of the OE substrate is a major determinant of transpososome mobility (Sakai et al 2000), we favor the idea that comigration of H-NS-bound forms of folded and unfolded transpososomes reflects H-NSdirected transpososome unfolding.…”

“…1; Sakai et al 1995). This bending permits transposase to contact the subterminal region of the OE, and in so doing, stabilizes the transposase-OE interaction-a critical step in transpososome formation (Sakai et al 2000;Crellin and Chalmers 2001). While precise details of how the two ends are brought together are not known, once formed, the Tn10 transpososome is quite stable and does not require the continued presence of IHF.…”

The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms

“…An alternative mechanism would be that direct interaction between IHF and transposase produces a conformational change in transposase that precludes target interactions. Limited support for a direct protein±protein interaction is provided by the observation that IHF has a higher af®nity for the IHF binding site in the Tn10 transpososome than for the free transposon end (19).…”

“…From the molecular model in Figure 1A, it is easy to visualize why Tn10 transposase would require a contribution from the energy of IHF binding or negative supercoiling to bend the DNA and establish the subterminal contacts. These contacts are required to activate the chemical steps during the cleavage reaction and must be disengaged to allow target interactions later in the reaction (18,19). In this paper, we investigate the symmetry of the IHF-folded arms of the transposon during transpososome assembly, and the properties of the conformational change leading to disengagement of the subterminal contacts and ejection of IHF from the complex.…”