Mendelman L V, Notarnicola S M, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10638-42. doi: 10.1073/pnas.89.22.10638.
The helicase and primase activities of bacteriophage T7 are distributed between the 56- and 63-kDa gene 4 proteins. The 56-kDa gene 4 protein lacks 63 amino acids found at the N terminus of the colinear 63-kDa protein and catalyzes helicase activity. The 63-kDa gene 4 protein catalyzes both primase and helicase activities. A bacteriophage deleted for gene 4, T7 delta 4-1, has been tested for growth by complementation on Escherichia coli strains that contain plasmids expressing either one or both of the gene 4 proteins. T7 delta 4-1 cannot grow (efficiency of plating, 10(-7)) on E. coli cells that express only 56-kDa gene 4 protein. In contrast, T7 delta 4-1 has an efficiency of plating of 0.1 on an E. coli strain that expresses only 63-kDa gene 4 protein in which glycine is substituted for methionine at position 64. A bacteriophage, T7 4B-, in which methionine at residue 64 is replaced by glycine, expresses only 63-kDa gene 4 protein. The burst sizes, latency periods, and Okazaki fragment sizes of T7 4B- are similar in the presence and absence of the 56-kDa gene 4 protein; however, T7 4B- has a reduced rate of DNA synthesis when compared with a phage that synthesizes both gene 4 proteins.
噬菌体T7的解旋酶和引发酶活性分布在56 kDa和63 kDa的基因4蛋白之间。56 kDa的基因4蛋白缺少共线性63 kDa蛋白N端的63个氨基酸，并催化解旋酶活性。63 kDa的基因4蛋白则同时催化引发酶和解旋酶活性。已通过在含有表达一种或两种基因4蛋白的质粒的大肠杆菌菌株上进行互补作用，对缺失基因4的噬菌体T7 delta 4-1的生长情况进行了测试。T7 delta 4-1在仅表达56 kDa基因4蛋白的大肠杆菌细胞上无法生长（平板效率为10^(-7)）。相比之下，T7 delta 4-1在仅表达63 kDa基因4蛋白（其中第64位的甘氨酸取代了甲硫氨酸）的大肠杆菌菌株上的平板效率为0.1。一种第64位残基的甲硫氨酸被甘氨酸取代的噬菌体T7 4B-，只表达63 kDa的基因4蛋白。在有和没有56 kDa基因4蛋白的情况下，T7 4B-的爆发量、潜伏期和冈崎片段大小相似；然而，与合成两种基因4蛋白的噬菌体相比，T7 4B-的DNA合成速率降低。
