Structural studies of bacteriophage genome packaging and ejection

描述（由申请人提供）：沙门氏菌感染噬菌体P22从dsDNA的串联体中包装其基因组。这种包装机械由大的（L-）和小的（S-）端酶复合物组成。小端酶的作用是通过pac位点正确定位大端酶进行包装起始。大端酶利用ATP水解将基因组的单个拷贝转移到噬菌体原衣壳中。l端酶的运动活性导致dsDNA的包装速度高达2000bp /秒。虽然人们对终止和裂解知之甚少，但在到达充满的原衣壳后，就会引起终止和裂解。切割后，L-/ s -末端酶复合体迅速与衣壳分离，使尾部和相关蛋白结合，将基因组密封在衣壳内。与沙门氏菌表面受体结合后，dsDNA连同射蛋白（gp7、gp16和gp20）被注入宿主细胞质。这些喷射蛋白是将dsDNA正确传递到宿主细胞质所必需的。目的1将[使用单粒子电子显微镜]阐明L-/S-终止酶全酶的三维结构和化学计量；与原衣壳结合之前的一种状态。[目前提出的终止酶机制是基于不确定的、低分辨率的34埃数据。优于7A的分辨率将解决该领域的机制争议。目的2将描述P22及其端酶复合物结合的三维结构；这是基因组包装之前的状态。Aim 3将捕获基因组结构和gp26（尾针）缺陷P22；这是一种刚刚经历了基因组喷射的状态。我们有gp26减噬菌体的初步[单颗粒]数据，显示尾针原来的密度增加了。我们的假设是，抛射（gp7、gp16或gp20）对这种密度有贡献。总之，这些目标将使新的假设得以发展，这将阐明基因组包装和注射到宿主体内的机制

DESCRIPTION (provided by applicant): The Salmonella-infecting bacteriophage P22 packages its genome from concatemers of dsDNA. This packaging machinery consists of a large (L-) and small (S-) terminase complex. The small terminase functions to properly position the large terminase for packaging initiation via the pac site. The large terminase uses ATP hydrolysis to translocate a single copy of the genome into the bacteriophage procapsid. The motor activity of L-terminase results in dsDNA packaging at a rate of up to 2000 bp/sec. Although poorly understood, termination and cleavage are induced upon reaching a filled procapsid. Upon cleavage, the L-/S-terminase complex quickly disassociates from the capsid enabling tail and associated proteins to bind, sealing the genome inside the procapsid. Upon binding to surface receptors of Salmonella, dsDNA along with ejection proteins (gp7, gp16, and gp20) are injected to host cytoplasm. These ejection proteins are required for correct delivery of dsDNA to host cytoplasm. Aim 1 will [use single particle electron microscopy] elucidate the 3D structure and stoichiometry of L-/S- terminase holoenzyme; a state just prior to binding to procapsid. [Current proposed mechanisms for terminase mechanism is based on inconclusive, low-resolution, 34 angstrom data. Resolution better than 7A will resolve mechanism controversies in the field.] Aim 2 will characterize the 3D structure of P22 with its terminase complex bound; a state just prior to genome packaging. Aim 3 will capture the structure of genome and gp26 (tail needle) deficient P22; a state just after genome ejection. We have preliminary [single particle] data of a gp26 minus phage that shows extra density where the tail needle used to be. It is our hypothesis that an ejection (gp7, gp16, or gp20) contributes to this density. Together, the aims will enable new hypotheses to be developed which will elucidate the mechanism of genome packaging and injection into the host