CRYOEM OF THE DECAMERIC RING FORMED BY THE P22 TERMINASE SMALL SUBUNIT (GP3)

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在许多噬菌体和一些真核病毒，如疱疹病毒和一些腺病毒的组装过程中，核酸通过一个独特的门状顶点插入到预先形成的衣壳前体中。在双链DNA病毒中，参与核酸包装的蛋白质复合体称为终止酶，通常由两个亚基组成。较小的亚基既负责识别病毒核酸中的唯一位点(PAC位点)，也负责启动终止酶复合体的形成。大亚基具有包装所需的酶活性，即在包装活动开始和结束时，为DNA转位提供能量的ATPase活性和切割DNA的内切酶活性。 终止酶复合体组装和DNA包装的分子机制对于任何病毒都不是很详细。末端酶亚单位的结构/功能关系的鉴定尤其难以捉摸。小亚基与其DNA识别位点的相互作用对于启动生产性包装和最终病毒成熟至关重要。小亚基也与大亚基结合，并刺激其ATPase活性。了解小亚基的三维结构将极大地促进我们对末端酶复合体组装和包装起始的理解。这些结构信息还将利用补充技术(拉曼光谱、CD光谱、沉积平衡等)加强对我们实验室收集的数据的解释。GP3的结构也将为拟议的未来涉及PAC位点的GP3/DNA结合的研究提供有价值的帮助。 在我们正在进行的对噬菌体P22的大小(GP2)终端酶亚基的研究中，我们已经制定了有效纯化两者的程序，使其同源性达到99%以上。我们最近发现，小亚基寡聚成一个对称的十聚体环，能够与dsDNA结合。我们已经通过平均负染色的透射电子显微图像来可视化了十角机。图1中显示的图像是通过使用与参考无关的方法和使用平移和旋转操作从~450个排列的粒子中精炼出来的，而不需要对排列做任何对称性假设。该环具有10重对称性，中心孔直径约2 nm，外部有10个尖峰。环的外径约为11.2 nm。 虽然已经提出了噬菌体SPP1和T4的小末端酶亚基的环形成，但寡聚体的状态一直是推测的，结构细节还没有揭示。对这些噬菌体的生物信息学和分子生物学实验也表明，小亚基在终止酶的组装和功能中扮演着重要的角色。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the assembly of many bacteriophages and some eukaryotic viruses, such as herpesviruses and some adenoviruses, the nucleic acid is inserted into a preformed capsid precursor through a unique portal vertex. In the double-stranded DNA viruses the protein complex actively involved in nucleic acid packaging is called terminase and usually consists of two subunits. The smaller subunit is responsible for both the recognition of a unique site in the viral nucleic acid (pac site) and the initiation of terminase complex formation. The large subunit possesses the necessary enzymatic activities for packaging, namely ATPase activity to provide energy for DNA translocation and endonuclease activity for cleavage of DNA at the beginning and end of the packaging event. Molecular mechanisms of terminase complex assembly and DNA packaging are not understood in detail for any virus. Identification of structure/function relationships for terminase subunits has been particularly elusive. Interaction of the small subunit with its DNA recognition site is crucial for the initiation of productive packaging and eventual virus maturation. The small subunit also binds to the large subunit and stimulates its ATPase activity. Knowledge of the three-dimensional structure of the small subunit would significantly advance our understanding of terminase complex assembly and initiation of packaging. Such structural information will also enhance the interpretation of data already collected in our laboratory using complementary techniques (Raman spectroscopy, CD spectroscopy, sedimentation equilibria, etc.). The structure of gp3 would also provide valuable assistance in proposed future investigations of gp3/DNA binding involving the pac site. In our ongoing studies of the small and large (gp2) terminase subunits of bacteriophage P22, we have developed procedures for efficient purification of both to better than 99% homogeneity. We have found recently that the small subunit oligomerizes into a symmetrical decameric ring that is able to bind to dsDNA. We have visualized the decamer from averaging negatively stained transmission electron micrographs. The image shown in Figure 1 was refined from ~450 aligned particles by use of a reference independent method and by using translation and rotation operations without any symmetry assumption for the alignment. The ring exhibits ten-fold symmetry with a central hole of ~2 nm diameter and ten outer spikes. The outer diameter of the ring is about 11.2 nm. Although ring formation has been proposed for small terminase subunits of bacteriophages SPP1 and T4, the oligomeric states have been speculative and no structural details have been revealed. Bioinformatics and molecular biology experiments on these phages also suggest important roles for the small subunit in terminase assembly and function.