title: Formation of a nucleoprotein complex containing Tn7 and its target DNA regulates transposition initiation.
abstract: Tn7 insertion into its specific target site, attTn7, is mediated by the proteins TnsA, TnsB, TnsC and TnsD. The double-strand breaks that separate Tn7 from the donor DNA require the Tns proteins, the transposon and an attTn7 target DNA, suggesting that a prerequisite for transposition is the formation of a nucleoprotein complex containing TnsABC+D, and these DNAs. Here, we identify a TnsABC+D transposon-attTn7 complex, and demonstrate that it is a transposition intermediate. We demonstrate that an interaction between TnsB, the transposase subunit that binds to the transposon ends, and TnsC, the target DNA-binding protein that controls the activity of the transposase, is essential for assembly of the TnsABC+D transposon-attTn7 complex. We also show that certain TnsB residues are required for recombination because they mediate a TnsB-TnsC interaction critical to formation of the TnsABC+D transposon-attTn7 complex. We demonstrate that TnsA, the other transposase subunit, which also interacts with TnsC, greatly stabilizes the TnsABC+D transposon-attTn7 complex. Thus multiple interactions between the transposase subunits, TnsA and TnsB, and the target-binding transposase activator, TnsC, control Tn7 transposition.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, USA.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Skelding
authors.forename: Robert
authors.initials: R
authors.lastname: Sarnovsky
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Craig
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D010583:Bacteriophage mu / Q000502:physiology; D004251:DNA Transposable Elements / Q000502:physiology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008274:Magnesium / Q000502:physiology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000737:chemistry*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009698:Nucleoproteins; C098908:TnsA protein, E coli; C069633:TnsB protein, E coli; C074940:TnsC protein, E coli; C462946:TnsD protein, human; D008274:Magnesium