MORPHOGENESIS OF BACTERIOPHAGE PHI-29

噬菌体 PHI-29 的形态发生

• 批准号: 3482724
• 负责人: DWIGHT ANDERSON
• 金额: $ 17.58万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 1994-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3482724
• 关键词: Bacillus subtilis; DNA replication; X ray crystallography; adenosine triphosphate; autoradiography; bacterial virus; binding proteins; conformation; gene expression; genetic manipulation; genetic mapping; histogenesis; image processing; microorganism growth; nucleic acid sequence; protein biosynthesis; radiotracer; temperature sensitive mutant; virus DNA; virus envelope; virus genetics; virus protein

A fully defined system for DNA packaging has been developed for Bacillus subtilis bacteriophage 029 that approximates in vivo assembly in efficiency. ATP, the DNa packaging protein gp16, purified proheads and DNA-gp3 comprise the complete system. A small RNA of 174 nucleotides is essential for DNA-gp3 packaging and is associated with the prohead vertex. The RNA can be detached and reattached, with concomitant loss and restoration of competence to package DNA-gp3 in the defined system. The gp16, overproduced in Escherichia coli, contains ATP-binding sequences, and the binding and hydrolysis of ATP by gp16 is both prohead and DNA-gp3 dependent. Approximately one molecule of ATP is used to package two base-pairs of 029 DNA. We have proposed a new model for prohead assembly in which RNa initiates assembly by binding the connector protein gp10 to form pentameric protomers. These protomers assemble into a particle by the addition of the scaffold and shell proteins. Further recruitment of shell protein occurs with the loss of gp10. The insertion of a connector dodecamer then forms the portal vertex and permits the final assembly of the neck- tail complex. The three-dimensional structure of the 029 prohead, packaging intermediates and the DNA-filled head will be studied by single crystal X-ray diffraction and by image analysis of electron micrographs of frozen-hydrated samples. These studies will complement our study of the morphopoietic properties of prohead RNA and the scaffold and connector proteins in head assembly. Purified RNa and prohead proteins and a number of unusual particles produced in nonpermissive infections by temperature-sensitive (ts) mutants will be used to confirm our assembly model and ultimately to construct the competent prohead in vitro. We will study the role of the shell protein (gp8) during DNA-gp3 packaging. We will study conformational changes of gp8, functional contact between the shell protein and DNA-gp3, and a direct role of gp8 that may involve ATP hydrolysis during DNA-gp3 packaging. We will study quantized packaging of DNA-gp3 by altering the packaging environment, by the use of proheads formed by ts8 mutants, and by the study of particles that package genomic fragments. We will investigate the hypothesis that RNA may add stability and orientation to the DNA- gp3 complex during packaging. We are studying prohead assembly, DNA encapsidation, and the mechanisms of form determination in the most efficient in vitro DNA packaging and viral assembly system known.

已开发出完全定义的 DNA 包装系统 近似体内的枯草芽孢杆菌噬菌体029 装配效率。 ATP，DNA 包装蛋白 gp16， 纯化的前头和 DNA-gp3 构成了完整的系统。 一个 174 个核苷酸的小 RNA 对于 DNA-gp3 包装至关重要 与前头顶点相关联。 RNA 可以被分离并 重新附着，同时丧失和恢复能力 将 DNA-gp3 封装在定义的系统中。 gp16，生产过剩 大肠杆菌，含有 ATP 结合序列，并且结合 gp16 对 ATP 的水解既是 prohead 又是 DNA-gp3 依赖。 大约用一分子 ATP 来包装 029 DNA 的两个碱基对。 我们提出了一个新模型 对于前头组装，RNA 通过结合 连接器蛋白 gp10 形成五聚体原聚体。 这些 通过添加支架，原聚体组装成颗粒 和壳蛋白。 壳蛋白的进一步募集发生 随着gp10的损失。 然后插入连接器十二角体 形成入口顶点并允许颈部的最终组装 尾部复合体。 029前头的三维结构， 包装中间体和 DNA 填充头将由 单晶 X 射线衍射和电子图像分析 冷冻水合样品的显微照片。 这些研究将 补充我们对前体RNA形态生成特性的研究 以及头部组装中的支架和连接器蛋白质。 纯化的 RNA 和前头蛋白以及产生的许多不寻常的颗粒 温度敏感（ts）突变体的非允许感染 将用于确认我们的装配模型并最终 体外构建主管前头。 我们将研究这个角色 DNA-gp3 包装过程中壳蛋白 (gp8) 的变化。 我们将学习 gp8的构象变化，壳之间的功能性接触 蛋白质和 DNA-gp3，以及可能涉及 ATP 的 gp8 的直接作用 DNA-gp3 包装过程中的水解。 我们将研究量子化 通过改变包装环境来包装DNA-gp3， 使用由 ts8 突变体形成的前头，并通过研究 包装基因组片段的颗粒。 我们将调查 假设 RNA 可以增加 DNA 的稳定性和方向性 包装过程中的 gp3 复合物。 我们正在研究前头组装， DNA 衣壳化和形态决定机制 最有效的体外 DNA 包装和病毒组装系统 已知。