title: 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.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Sakai
authors.forename: N
authors.initials: N
authors.lastname: Kleckner
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.forename: A
authors.initials: A
authors.lastname: Guhathakurta
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D034982:Integration Host Factors; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D003850:Deoxyribonuclease I