Membrane-associated assembly of a phage T4 DNA entrance vertex structure studied with expression vectors.
Membrane-associated assembly of a phage T4 DNA entrance vertex structure studied with expression vectors.
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DOI：
10.1016/0022-2836(89)90599-8
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发表时间：
1989-10
影响因子：
5.6
通讯作者：
G. Michaud;A. Zachary;V. Rao;L. Black
中科院分区：
文献类型：
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作者：
G. Michaud;A. Zachary;V. Rao;L. Black
噬菌体头的DNA入口顶点对前体组装和DNA包装至关重要。单个结构蛋白包含这种前体的十二聚体环亚结构。噬菌体T4前体的组装发生在细胞质膜上，通过gp20 DNA入口顶点或附近的特定附着。一个辅助的头部组装基因产物gp40被假设参与gp20亚结构的组装。在λpL启动子控制下，将T4基因20、40和20+40克隆到表达载体上。相应的T4基因产物是高产合成的，通过对体内相应的感染T4突变体的补体能力判断其具有活性。将克隆的T4基因20和基因40作为整体膜蛋白插入细胞质膜；然而，只有在gp40合成的情况下，gp20才会插入到膜中，而gp40在gp20存在或不存在的情况下都可以插入。gp20的插入需要膜电位，不依赖于大肠杆菌基因，并且根据膜保护的特定蛋白酶片段判断，具有确定的跨膜构象。通过抗体结合和蛋白a -金免疫电镜可以探测插入的gp20结构。这些数据表明，一个特定的gp20-gp40膜插入结构构成了T4前体组装起始复合物。
The DNA entrance vertex of the phage head is critical for prohead assembly and DNA packaging. A single structural protein comprises this dodecameric ring substructure of the prohead. Assembly of the phage T4 prohead occurs on the cytoplasmic membrane through a specific attachment at or near the gp20 DNA entrance vertex. An auxiliary head assembly gene product, gp40, was hypothesized to be involved in assembling the gp20 substructure. T4 genes20, 40and20+40were cloned into expression vectors under λpL promoter control. The corresponding T4 gene products were synthesized in high yield and were active as judged by their ability to complement the corresponding infecting T4 mutantsin vivo. The cloned T4 gene20and gene40products were inserted into the cytoplasmic membrane as integral membrane proteins; however, gp20 was inserted into the membrane only when gp40 was also synthesized, whereas gp40 was inserted in the presence or absence of gp20. The gp20 insertion required a membrane potential, was not dependent upon theEscherichia coli groEgene, and assumed a defined membrane-spanning conformation, as judged by specific protease fragments protected by the membrane. The inserted gp20 structure could be probed by antibody binding and protein A—gold immunoelectron microscopy. The data suggest that a specific gp20-gp40-membrane insertion structure constitutes the T4 prohead assembly initiation complex.