Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 2013 May 28;110(22):E2038-45. doi: 10.1073/pnas.1305716110. Epub 2013 May 14.
The transposon Tn7 transposase that recognizes the transposon ends and mediates breakage and joining is heteromeric. It contains the Tn7-encoded proteins TnsB, which binds specifically to the transposon ends and carries out breakage and joining at the 3' ends, and TnsA, which carries out breakage at the 5' ends of Tn7. TnsA apparently does not bind specifically to DNA, and we have hypothesized that it is recruited to the ends by interaction with TnsB. In this work, we show that TnsA and TnsB interact directly and identify several TnsA and TnsB amino acids involved in this interaction. We also show that TnsA can stimulate two key activities of TnsB, specific binding to the ends and pairing of the Tn7 ends. The ends of Tn7 are structurally asymmetric (i.e., contain different numbers of TnsB-binding sites), and Tn7 also is functionally asymmetric, inserting into its specific target site, attachment site attTn7 (attTn7) in a single orientation. Moreover, Tn7 elements containing two Tn7 right ends can transpose, but elements with two Tn7 left ends cannot. We show here that TnsA + TnsB are unable to pair the ends of a Tn7 element containing two Tn7 left ends. This pairing defect likely contributes to the inability of Tn7 elements with two Tn7 left ends to transpose.
转座子 Tn7 转座酶识别转座子末端并介导断裂和连接是异源的。它包含 Tn7 编码的蛋白 TnsB，它特异性地结合转座子末端，并在 3'末端进行断裂和连接，以及 TnsA，它在 Tn7 的 5'末端进行断裂。TnsA 显然不特异性地结合 DNA，我们假设它通过与 TnsB 的相互作用被募集到末端。在这项工作中，我们表明 TnsA 和 TnsB 直接相互作用，并确定了参与这种相互作用的几个 TnsA 和 TnsB 氨基酸。我们还表明 TnsA 可以刺激 TnsB 的两个关键活性，即特异性结合末端和 Tn7 末端的配对。Tn7 的末端结构不对称（即，含有不同数量的 TnsB 结合位点），并且 Tn7 也具有功能不对称性，以单一取向插入其特定靶位点，附着位点 attTn7（attTn7）。此外，含有两个 Tn7 右末端的 Tn7 元件可以转座，但含有两个 Tn7 左末端的元件不能。我们在这里表明，TnsA+TnsB 无法配对含有两个 Tn7 左末端的 Tn7 元件的末端。这种配对缺陷可能导致含有两个 Tn7 左末端的 Tn7 元件无法转座。
