MORPHOGENESIS OF BACTERIOPHAGE PHI-29

一个完全定义的DNA包装系统已经被开发出来 枯草杆菌噬菌体029，与体内相似 高效组装。ATP，DNA包装蛋白gp16， 纯化的蛋白质和DNA-GP3组成了完整的体系。一个 174个核苷酸的小RNA是DNA-GP3包装和 与前部顶点相关联。RNA可以被分离并 重新连接，随之而来的是丧失和恢复能力 将DNA-GP3打包到定义的系统中。Gp16，生产过剩于 大肠杆菌，含有三磷酸腺苷结合序列，并结合 Gp16对三磷酸腺苷的水解率和DNA-gp3的同源性。 依附的。大约一个三磷酸腺苷分子被用来包装 两个029DNA的碱基对。我们提出了一种新的模式 对于前头组件，其中RNA通过结合 连接蛋白GP10形成五聚体原核。这些 通过添加支架，原形分子组装成颗粒 和壳蛋白。壳蛋白的进一步招募发生 随着GP10的丧失。然后插入连接器十二聚体 形成入口顶点，并允许颈部的最终组装- 尾巴情结。029引线的三维结构， 包装中间体和充满DNA的头部将通过 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蛋白。纯净的 RNA和前部蛋白质以及一些不寻常的颗粒产生 温度敏感(Ts)突变体的非允许性感染 将用于确认我们的组装模型，并最终 体外构建有竞争力的探针。我们将研究这一角色 壳蛋白(GP8)在DNA-GP3包装过程中的变化。我们会研究 GP8的构象变化，壳与壳之间的功能接触 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，对DNA-GP3进行包装 利用ts8突变体形成的探针，并通过对 包装基因组片段的颗粒。我们将调查 假设RNA可以增加DNA的稳定性和定向性- 包装过程中的GP3复合体。我们正在研究前置组件， DNA的包埋和形态确定的机制 最高效的体外DNA包装和病毒组装系统 为人所知。

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.