Structural basis of phage infection and DNA ejection

项目摘要/摘要 噬菌体是生物圈中最丰富的生物实体，是许多细菌的来源 进化论。大多数噬菌体利用复杂的尾部机器将它们的病毒DNA和蛋白质转移到宿主细胞中。 在上一次赠款期间，我们在记录几个 经典噬菌体(T7、T4、P22、Sp6、Φ29和λ)。我们的研究为这种机制提供了新的分子见解。 通过这些尾巴噬菌体克服细菌细胞膜的多重障碍，并将它们的 遗传物质进入宿主细胞细胞质。这里我们完全集中在T7上，因为一个完整的机械 T7感染是如何开始的，DNA易位是如何影响的，目前仍不清楚。该信号 触发蛋白质喷射的机制尚不清楚。核心蛋白是如何穿透外膜的？ 细胞膜破裂？加长的尾巴纳米机器如何工作来运送它的货物- 噬菌体基因组--进入细胞？同样重要的是，尾巴和纤维的构象变化是怎样的 协调，以至于噬菌体与敏感细胞的最初相互作用几乎不可阻挡地导致感染？ 对任何噬菌体系统来说，一个完全意想不到的观察是，T7在 感染的开始。我们的中心假设是T7经历了巨大的构象变化 促进吸附、通道形成和DNA移位。我们的协作实验方法 原位结构生物学与遗传学密切相关，生理学将解决这些基本问题 问题。三个具体目标是：(1)剖析T7基因组喷射机的结构和功能； (2)确定延长的T7尾巴如何跨越细胞膜；(3)阐明吸附和 F1FO三磷酸腺苷合成酶的招募。

PROJECT SUMMARY/ABSTRACT Bacteriophages are the most abundant biological entity in the biosphere and are responsible for much of bacterial evolution. Most phages utilize elaborate tail machines to translocate their viral DNA and proteins into a host cell. During the last grant period, we made considerable progress in documenting infection initiation from several classic phages (T7, T4, P22, SP6, Φ29, and λ). Our studies provided new molecular insights into the mechanisms by which these tailed phages overcome the multiple barriers of the bacterial cell envelope and to deliver their genetic material into the host cell cytoplasm. Here we focus entirely on T7, because a complete mechanistic description of how T7 infection is initiated and how DNA translocation is effected remains elusive. The signal triggering protein ejection is not known. How do the core proteins penetrate the outer membrane, and how is the cytoplasmic membrane breached? How does the extended tail nanomachine function to deliver its cargo – the phage genome – into a cell? Equally importantly, how are the conformational changes in the tail and fibers coordinated so that the initial interaction of a phage with a susceptible cell almost inexorably leads to infection? A completely unanticipated observation for any phage system is that T7 recruits the host F1FO ATP synthase at the initiation of infection. Our central hypothesis is that T7 undergoes massive conformational changes to facilitate adsorption, channel formation, and DNA translocation. Our collaborative experimental approach where structural biology in situ is intimately coupled to genetics and physiology will address these fundamental questions. Three specific aims are: (1) Dissect the structure and function of the T7 genome ejection machine; (2) determine how the extended T7 tail spans the cell envelope; (3) illuminate mechanisms of adsorption and recruitment of the F1FO ATP synthase.