Formation of a nucleoprotein complex containing Tn7 and its target DNA regulates transposition initiation.
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.
Skelding
Zachary
Z
Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Sarnovsky
Robert
R
Craig
Nancy L
NL
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
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