Protein-DNA contacts and conformational changes in the Tn10 transpososome during assembly and activation for cleavage

IHF or supercoiling is required early in TWO transposition, but at later st ages they inhibit the reaction in a classic homeostatic loop. We investigat ed the mechanism of transpososome assembly and regulation using hydroxyl ra dical DNA protection and interference. We present a three-dimensional molec ular model for the IHF-bent end of Tn10 wrapped around a transposase core. Contacts span some 80 bp at the transposon end, but after assembly of an ac tive complex containing metal ion, most contacts become dispensable. These include transposase contacts beyond the IHF site that chaperone assembly of the complex and are needed for efficient cleavage. Single and double-end b reaks do not affect the complex but divalent metal ions promote large confo rmational changes at bp +1 and the flanking DNA.