Howard Hughes Medical Institute andDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Howard Hughes Medical Institute andDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205
Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):E2858-65. doi: 10.1073/pnas.1409869111. Epub 2014 Jun 30.
The excision of transposon Tn7 from a donor site and its insertion into its preferred target site, attachment site attTn7, is mediated by four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC, and TnsD. Transposition requires the assembly of a nucleoprotein complex containing all four Tns proteins and the DNA substrates, the donor site containing Tn7, and the preferred target site attTn7. TnsA and TnsB together form the heteromeric Tn7 transposase, and TnsD is a target-selecting protein that binds specifically to attTn7. TnsC is the key regulator of transposition, interacting with both the TnsAB transposase and TnsD-attTn7. We show here that TnsC interacts directly with TnsB, and identify the specific region of TnsC involved in the TnsB-TnsC interaction during transposition. We also show that a TnsC mutant defective in interaction with TnsB is defective for Tn7 transposition both in vitro and in vivo. Tn7 displays cis-acting target immunity, which blocks Tn7 insertion into a target DNA that already contains Tn7. We provide evidence that the direct TnsB-TnsC interaction that we have identified also mediates cis-acting Tn7 target immunity. We also show that TnsC interacts directly with the target selector protein TnsD.
转座子 Tn7 从供体位点的切除及其插入到其优选靶位 attTn7 由四个 Tn7 编码的转位蛋白：TnsA、TnsB、TnsC 和 TnsD 介导。转位需要组装一个包含所有四个 Tns 蛋白和 DNA 底物的核蛋白复合物，供体位点含有 Tn7，而优选靶位 attTn7。TnsA 和 TnsB 一起形成异源 Tn7 转位酶，而 TnsD 是一种特异性结合 attTn7 的靶位选择蛋白。TnsC 是转位的关键调节剂，与 TnsAB 转位酶和 TnsD-attTn7 相互作用。我们在这里表明，TnsC 与 TnsB 直接相互作用，并确定了转位过程中 TnsC 与 TnsB 相互作用所涉及的特定区域。我们还表明，与 TnsB 相互作用缺陷的 TnsC 突变体在体外和体内均不能进行 Tn7 转位。Tn7 表现出顺式作用靶位免疫，这阻止了 Tn7 插入已经含有 Tn7 的靶 DNA。我们提供的证据表明，我们鉴定的直接 TnsB-TnsC 相互作用也介导顺式作用的 Tn7 靶位免疫。我们还表明，TnsC 与靶位选择蛋白 TnsD 直接相互作用。
