Department of Biology, The Catholic University of America, Washington, DC 20064, USA.
Proc Natl Acad Sci U S A. 2012 Dec 4;109(49):20000-5. doi: 10.1073/pnas.1209214109. Epub 2012 Nov 19.
Tailed bacteriophages and herpes viruses use powerful ATP-driven molecular motors to translocate their viral genomes into a preformed capsid shell. The bacteriophage T4 motor, a pentamer of the large terminase protein (gp17) assembled at the portal vertex of the prohead, is the fastest and most powerful known, consistent with the need to package a ~170-kb viral genome in approximately 5 min. Although much is known about the mechanism of DNA translocation, very little is known about how ATP modulates motor-DNA interactions. Here, we report single-molecule measurements of the phage T4 gp17 motor by using dual-trap optical tweezers under different conditions of perturbation. Unexpectedly, the motor pauses randomly when ATP is limiting, for an average of 1 s, and then resumes translocation. During pausing, DNA is unpackaged, a phenomenon so far observed only in T4, where some of the packaged DNA is slowly released. We propose that the motor pauses whenever it encounters a subunit in the apo state with the DNA bound weakly and incorrectly. Pausing allows the subunit to capture ATP, whereas unpackaging allows scanning of DNA until a correct registry is established. Thus, the "pause-unpackaging" state is an off-translocation recovery state wherein the motor, sometimes by taking a few steps backward, can bypass the impediments encountered along the translocation path. These results lead to a four-state mechanochemical model that provides insights into the mechanisms of translocation of an intricately branched concatemeric viral genome.
长尾噬菌体和疱疹病毒利用强大的 ATP 驱动分子马达将其病毒基因组转运到预先形成的衣壳壳中。噬菌体 T4 马达是五聚体的大型末端酶蛋白（gp17），组装在前头的门顶点，是已知最快和最强大的马达，这与需要在大约 5 分钟内包装一个约 170kb 的病毒基因组一致。尽管人们对 DNA 转运的机制有了很多了解，但对于 ATP 如何调节马达-DNA 相互作用却知之甚少。在这里，我们通过使用双阱光镊在不同的扰动条件下对噬菌体 T4 gp17 马达进行了单分子测量。出乎意料的是，当 ATP 有限时，马达会随机暂停，平均暂停 1 秒，然后恢复转运。在暂停期间，DNA 会解包，这种现象迄今为止只在 T4 中观察到，其中一些包装的 DNA 会缓慢释放。我们提出，当马达遇到结合较弱和不正确的 DNA 的apo 状态的亚基时，它会暂停。暂停允许亚基捕获 ATP，而解包允许 DNA 扫描，直到建立正确的注册。因此，“暂停-解包”状态是一种非转运恢复状态，其中马达有时可以通过向后走几步，绕过在转运路径中遇到的障碍。这些结果导致了一个四态机械化学模型，该模型提供了对复杂分支串联病毒基因组转运机制的深入了解。
