TY - JOUR
T1 - Architecture of the Tn7 posttransposition complex
T2 - An elaborate nucleoprotein structure
AU - Holder, Jason W.
AU - Craig, Nancy L.
N1 - Funding Information:
We thank Robert Sarnovsky for cloning of TnsC into pCYB1; Prasad Kuduvalli, Gregory McKenzie, Chuck Merryman, and Rupak Mitra for helpful discussions; Jan Hoh for assistance with AFM; and Iva Ivanovska, Helen McComas, and Patti Kodeck for assistance with manuscript preparation. J.W.H. and N.L.C. conceived of and designed the experiments. J.W.H. performed the experiments and J.W.H. and N.L.C. wrote the paper. This work was supported by NIH grants P01 CA16519 and RO1 GM076425 to N.L.C. and NIH Training Grant T32-GM07445 to the Biochemistry, Cellular and Molecular Biology Program at Johns Hopkins University. N.L.C. is an investigator of the Howard Hughes Medical Institute. All authors declare no actual or potential conflict of interest.
PY - 2010/8
Y1 - 2010/8
N2 - Four transposition proteins encoded by the bacterial transposon Tn7, TnsA, TnsB, TnsC, and TnsD, mediate its site- and orientation-specific insertion into the chromosomal site attTn7. To establish which Tns proteins are actually present in the transpososome that executes DNA breakage and joining, we have determined the proteins present in the nucleoprotein product of transposition, the posttransposition complex (PTC), using fluorescently labeled Tns proteins. All four required Tns proteins are present in the PTC in which we also find that the Tn7 ends are paired by protein-protein contacts between Tns proteins bound to the ends. Quantification of the relative amounts of the fluorescent Tns proteins in the PTC indicates that oligomers of TnsA, TnsB, and TnsC mediate Tn7 transposition. High-resolution DNA footprinting of the DNA product of transposition attTn7::Tn7 revealed that about 350 bp of DNA on the transposon ends and on attTn7 contact the Tns proteins. All seven binding sites for TnsB, the component of the transposase that specifically binds the ends and mediates 3' end breakage and joining, are occupied in the PTC. However, the protection pattern of the sites closest to the Tn7 ends in the PTC are different from that observed with TnsB alone, likely reflecting the pairing of the ends and their interaction with the target nucleoprotein complex necessary for activation of the breakage and joining steps. We also observe extensive protection of the attTn7 sequences in the PTC and that alternative DNA structures in substrate attTn7 that are imposed by TnsD are maintained in the PTC.
AB - Four transposition proteins encoded by the bacterial transposon Tn7, TnsA, TnsB, TnsC, and TnsD, mediate its site- and orientation-specific insertion into the chromosomal site attTn7. To establish which Tns proteins are actually present in the transpososome that executes DNA breakage and joining, we have determined the proteins present in the nucleoprotein product of transposition, the posttransposition complex (PTC), using fluorescently labeled Tns proteins. All four required Tns proteins are present in the PTC in which we also find that the Tn7 ends are paired by protein-protein contacts between Tns proteins bound to the ends. Quantification of the relative amounts of the fluorescent Tns proteins in the PTC indicates that oligomers of TnsA, TnsB, and TnsC mediate Tn7 transposition. High-resolution DNA footprinting of the DNA product of transposition attTn7::Tn7 revealed that about 350 bp of DNA on the transposon ends and on attTn7 contact the Tns proteins. All seven binding sites for TnsB, the component of the transposase that specifically binds the ends and mediates 3' end breakage and joining, are occupied in the PTC. However, the protection pattern of the sites closest to the Tn7 ends in the PTC are different from that observed with TnsB alone, likely reflecting the pairing of the ends and their interaction with the target nucleoprotein complex necessary for activation of the breakage and joining steps. We also observe extensive protection of the attTn7 sequences in the PTC and that alternative DNA structures in substrate attTn7 that are imposed by TnsD are maintained in the PTC.
KW - DNA footprinting
KW - Expressed protein ligation
KW - Nucleoprotein complex
KW - Transposase
KW - Transposition
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U2 - 10.1016/j.jmb.2010.06.003
DO - 10.1016/j.jmb.2010.06.003
M3 - Article
C2 - 20538004
AN - SCOPUS:77955087046
SN - 0022-2836
VL - 401
SP - 167
EP - 181
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 2
ER -