Morphogenesis of Bacteriophage T4

在过去三年的NSF支持期间，在理解利用噬菌体T4对病毒组装的形状和大小的调节方面取得了进展。对头部长度调节的研究表明，病毒内部的DNA被选择以特定的长度包装，这可以参考二十面体表面晶格理论来解释。因此，T4中的DNA包装取决于衣壳在设置其长度参数时所使用的设计原则。利用DNA的脉冲场电泳法为分离T4DNA分子提供了迄今为止最高的分辨率。这里提出的新工作将集中在确定DNA包装的测量系统如何运作上。噬菌体T4的头部是拉长的，而不是大多数病毒的二十面体。内部蛋白核心或支架蛋白的突变都会导致头部恢复到二十面体的形式。所有导致头部拉长的突变(称为巨人)都位于编码病毒外壳蛋白(GP 23)的基因中。我们将通过克隆表达载体中支架和外壳蛋白的基因，定点缺失和突变，以及分子结构确定，来测试GP 23单独控制头部伸长的想法。提纯的gp23将在结晶条件下进行X射线结构测定。在已提出的四个确定T4头部长度的模型中，有两个被认为是可以消除的(简单模板模型和动力学模型)。这将扩展并证实初步数据，这些数据表明长度是由双组分蛋白质游标机制和二十面体顶点累积应变的稳定相结合控制的。总之，拟议的实验将在很大程度上有助于理解形状和大小控制的机制，以及亚细胞水平的DNA包装。

During the past three years of NSF support, progress was made in understanding the regulation of shape and size in virus assembly using bacteriophage T4. Studies on head length regulation have shown that the DNA inside the virus is selected for packaging at specific lengths that can be interpreted by reference to icosahedral surface lattice theory. DNA packaging in T4 is therefore dependent on the design principles used by the capsid in setting its length parameters. Use of pulsed-field electrophoresis of DNA has provided the highest resolution yet for the separation of T4 DNA molecules. The new work proposed here will focus on how the measuring system that determines DNA packaging operates. The head of phage T4 is elongated, not icosahedral as for most viruses. Mutations in the internal protein core or scaffold proteins all cause the head to revert to its icosahedral form. All mutations that caused elongated heads (called giants) are located in the gene coding for the virus coat protein (gp 23). We will test the idea that gp 23 alone controls head elongation using cloned genes for both the scaffold and the coat protein in expression vectors, site-directed deletions and mutagenesis, and molecular structure determination. Purified gp23 will be subjected to crystallization conditions for x-ray structure determination. Of the four models for T4 head length determination that have been proposed, it is believed two can be eliminated (the simple template and the kinetic models). This will now extend and confirm preliminary data that suggest that length is controlled by a combination of a two-component protein vernier mechanism and stabilization of accumulated strain at the icosahedral vertices. Together, the proposed experiments will help substantially in understanding of the mechanisms of shape and size control, and DNA packaging, at the subcellular level.