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

P22 末端酶小亚基 (GP3) 形成的十聚环的冷冻

• 批准号: 7723577
• 负责人: GEORGE THOMAS
• 金额: $ 1.26万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723577
• 关键词: ATP phosphohydrolase; Adenoviruses; Bacteriophage P22; Bacteriophages; Binding; Bioinformatics; Caliber; Capsid; Complex; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; DNA; DNA Binding; DNA Packaging; Data; Double Stranded DNA Virus; Electrons; Event; Exhibits; Funding; Future; Grant; Herpesviridae; Image; Institution; Investigation; Knowledge; Laboratories; Methods; Molecular; Molecular Biology; Nucleic Acids; Operative Surgical Procedures; Procedures; Raman Spectrum Analysis; Research; Research Personnel; Resources; Role; Rotation; Simplexvirus; Site; Source; Spectrum Analysis; Staining method; Stains; Structure; Structure-Activity Relationship; Techniques; Translations; United States National Institutes of Health; Viral; Viral Proteins; Virus; endonuclease; particle; research study; sedimentation equilibrium; terminase; three dimensional structure; transmission process

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 在许多噬菌体和一些真核病毒（例如疱疹病毒和一些腺病毒）的组装过程中，核酸通过独特的入口顶点插入到预先形成的衣壳前体中。 在双链DNA病毒中，积极参与核酸包装的蛋白质复合物称为终止酶，通常由两个亚基组成。较小的亚基负责识别病毒核酸中的独特位点（pac位点）和启动终止酶复合物形成。 大亚基具有包装所需的酶活性，即为 DNA 易位提供能量的 ATP 酶活性和在包装事件开始和结束时为 DNA 裂解提供能量的核酸内切酶活性。 任何病毒的终止酶复合物组装和 DNA 包装的分子机制尚不清楚。 终止酶亚基的结构/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有效包装的启动和最终病毒的成熟至关重要。 小亚基还与大亚基结合并刺激其 ATP 酶活性。 了解小亚基的三维结构将显着增进我们对终止酶复合物组装和包装起始的理解。 此类结构信息还将增强对我们实验室使用补充技术（拉曼光谱、CD 光谱、沉降平衡等）收集的数据的解释。 gp3 的结构也将为未来涉及 pac 位点的 gp3/DNA 结合研究提供宝贵的帮助。 在我们对噬菌体 P22 的小和大 (gp2) 终止酶亚基正在进行的研究中，我们开发了有效纯化两者的程序，其同质性优于 99%。 我们最近发现小亚基寡聚成对称的十聚环，能够与双链DNA结合。 我们通过平均负染透射电子显微照片可视化了十聚体。图 1 中所示的图像是通过使用独立于参考的方法并使用平移和旋转操作从大约 450 个对齐的粒子中精炼出来的，而没有任何对齐的对称性假设。 该环表现出十重对称性，中心孔直径约为 2 nm，外部有十个尖峰。 环的外径约为11.2 nm。 尽管已提出噬菌体 SPP1 和 T4 的小末端酶亚基形成环，但寡聚状态一直是推测性的，并且尚未揭示结构细节。 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组装和功能中发挥重要作用。