Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore MD 21205, USA.
Current Address: Verenium Corporation. 4955 Directors Place, San Diego, CA 92121, USA.
Mob DNA. 2010 Jul 23;1(1):18. doi: 10.1186/1759-8753-1-18.
The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7, and insert just downstream of the highly conserved chromosomal glmS gene. TnsD is one of four transposon-encoded polypeptides (TnsABC+D) required for site-specific insertion of Tn7 into attTn7, and is the target site-selector that binds to a highly conserved sequence in the end of the glmS protein coding region. In this study, we identified important nucleotides within this region that are crucial for TnsD-attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. A key role of TnsD is to recruit the non-sequence specific DNA-binding protein TnsC to attTn7; TnsC also interacts with and controls both the TnsA and TnsB subunits of the Tn7 transposase. TnsC stimulates the binding of TnsD to attTn7 in vivo, and TnsCD and TnsD can also interact in the absence of DNA and localize their interaction domains to the N-terminal region of each protein.
细菌转座子 Tn7 的特点是能够识别一个称为 attTn7 的特定位点，并在高度保守的染色体 glmS 基因下游插入。TnsD 是 Tn7 特异性插入 attTn7 所需的四个转座子编码多肽（TnsABC+D）之一，是与 glmS 蛋白编码区末端高度保守序列结合的靶位点选择器。在这项研究中，我们确定了该区域内对 TnsD-attTn7 相互作用至关重要的重要核苷酸。我们还探测了与 attTn7 相互作用的 TnsD 区域，发现整个蛋白长度上都存在重要的 DNA 结合决定因素，包括一个氨基末端 CCCH 锌指模体。TnsD 的一个关键作用是将非序列特异性 DNA 结合蛋白 TnsC 募集到 attTn7 上；TnsC 还与 Tn7 转座酶的 TnsA 和 TnsB 亚基相互作用并控制它们。TnsC 刺激 TnsD 在体内与 attTn7 的结合，并且 TnsCD 和 TnsD 也可以在没有 DNA 的情况下相互作用，并将它们的相互作用域定位到每个蛋白质的氨基末端区域。
