Mechanism of ATP-Driven DNA Packaging in Bacteriophage T4

双链DNA病毒将DNA转移到预先形成的病毒外壳中，并将其组织成具有与DNA晶体一样高密度的结构的机制是一个迷人的，未解决的生物学问题。 在噬菌体T4中，E.在大肠杆菌裂解病毒中，56 mm长的DNA分子被包装在约0.115 mm长和0.085 mm宽的二十面体衣壳内。遗传和生物化学研究表明，一个ATP供电的DNA包装“机器”构成的prohead门户蛋白和两个非结构包装/终止酶蛋白驱动DNA包装。 该项目解决了DNA包装问题的核心问题之一，即，包装机的哪个部件消耗ATP并将其与DNA易位偶联？ 据推测，具有两个共有ATP结合位点并具有ATP酶活性的大末端酶亚基gp 17是噬菌体T4中的易位ATP酶。 这一假设将通过一种新的组合诱变范例进行测试。 将在gp 17中共有ATP结合位点的特征残基处构建由所有可能的密码子组合组成的突变体文库。 突变将通过位点特异性标记拯救策略转移到噬菌体T4基因组中。 将产生一系列表现出无效、噬斑大小和温度敏感表型的新型突变体。 将通过分子和生物化学方法分析选定的突变体，以测试推定的ATP酶中心和DNA易位之间的联系。 还将测试DNA移位旋转模型的预测。这些方法广泛适用于分析其他末端酶和ATP触发的生化机器中的功能位点，并作为培训本科生和研究生的一个令人兴奋的模型。

The mechanism by which double stranded DNA viruses translocate DNA into a preformed virus coat and organize it into a structure that has a density as high as a DNA crystal is a fascinating, unsolved, biological problem. In bacteriophage T4, an E. coli lytic virus, a 56 mm long DNA molecule is packaged within an icosahedral capsid that is approximately 0.115 mm long and 0.085 mm wide. Genetic and biochemical studies suggest that an ATP-powered DNA packaging "machine" constituted by the prohead portal protein and two nonstructural packaging/terminase proteins drives DNA packaging. This project addresses one of the central questions of the DNA packaging problem, i.e., which component of the packaging machine consumes the ATP and couples it to DNA translocation? It is hypothesized that the large terminase subunit gp17, which possesses two consensus ATP binding sites and exhibits an ATPase activity, is the translocating ATPase in phage T4. This hypothesis will be tested by a novel combinatorial mutagenesis paradigm. Mutant libraries consisting of all possible codon combinations will be constructed at the signature residues of the consensus ATP binding sites in gp17. The mutations will be transferred into phage T4 genome by a site-specific marker rescue strategy. A collection of novel mutants exhibiting null, plaque-size, and temperature sensitive phenotypes will be generated. Selected mutants will be analyzed by molecular and biochemical approaches to test the linkage between the putative ATPase centers and DNA translocation. The predictions of a rotatory model for DNA translocation will also be tested. These approaches are broadly applicable to analyze functional sites in other terminases and ATP-triggered biochemical machines, and serve as an exciting model for training undergraduate and graduate students.