Skelding Zachary, Sarnovsky Robert, Craig Nancy L
Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD, USA.
EMBO J. 2002 Jul 1;21(13):3494-504. doi: 10.1093/emboj/cdf347.
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.
Tn7插入其特定靶位点attTn7是由TnsA、TnsB、TnsC和TnsD蛋白介导的。使Tn7与供体DNA分离的双链断裂需要Tns蛋白、转座子和attTn7靶DNA，这表明转座的一个先决条件是形成包含TnsABC+D和这些DNA的核蛋白复合物。在这里，我们鉴定出了TnsABC+D转座子-attTn7复合物，并证明它是一个转座中间体。我们证明，与转座子末端结合的转座酶亚基TnsB和控制转座酶活性的靶DNA结合蛋白TnsC之间的相互作用对于TnsABC+D转座子-attTn7复合物的组装至关重要。我们还表明，某些TnsB残基对于重组是必需的，因为它们介导了对TnsABC+D转座子-attTn7复合物形成至关重要的TnsB-TnsC相互作用。我们证明，同样与TnsC相互作用的另一个转座酶亚基TnsA极大地稳定了TnsABC+D转座子-attTn7复合物。因此，转座酶亚基TnsA和TnsB与靶结合转座酶激活剂TnsC之间的多种相互作用控制着Tn7转座。
