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: 7956442
• 负责人: GEORGE THOMAS
• 金额: $ 1.29万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956442
• 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; Microscopy; Molecular; Molecular Biology; Nucleic Acids; Operative Surgical Procedures; Procedures; Raman Spectrum Analysis; Research; Research Personnel; Resources; Role; Rotation; 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; protein complex; 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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，核酸通过独特的门户顶点插入预先形成的衣壳前体中。 在双链DNA病毒中，活跃参与核酸包装的蛋白质复合物称为末端酶，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）和启动末端酶复合物的形成。 大亚基具有包装所必需的酶活性，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切割DNA的核酸内切酶活性。 对于任何病毒，末端酶复合物组装和DNA包装的分子机制都没有详细了解。 终止酶亚基的结构/功能关系的鉴定一直特别难以捉摸。 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ATP酶活性。 小亚基的三维结构的知识将显着推进我们对终止酶复合物组装和包装起始的理解。 这些结构信息也将增强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。 gp 3的结构也将提供宝贵的援助，在未来的调查建议gp 3/DNA结合涉及的PAC网站。 在我们正在进行的噬菌体P22的小和大（gp 2）末端酶亚基的研究中，我们已经开发出了有效纯化的程序，两者都优于99%的同质性。 我们最近发现，小亚基寡聚成一个对称的十聚体环，能够结合双链DNA。 我们已经可视化的十聚体平均负染色透射电子显微镜照片。图1中所示的图像是通过使用参考独立方法并通过使用平移和旋转操作从约450个对齐的颗粒中提炼出来的，而没有任何对齐的对称性假设。 该环具有十重对称性，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。 虽然环的形成已被提出为小的噬菌体SPP 1和T4的末端酶亚基，寡聚状态一直是推测性的，没有结构细节已被揭示。 生物信息学和分子生物学实验表明，这些小亚基在末端酶组装和功能中也发挥着重要作用。