Nakai H, Richardson C C
J Biol Chem. 1986 Nov 15;261(32):15217-24.
Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.
噬菌体T7的基因4蛋白和DNA聚合酶催化在单链DNA模板上以RNA为引物的DNA合成。T7 DNA聚合酶对基因4蛋白和单链DNA均表现出亲和力，并且在dTTP存在的情况下，基因4蛋白能稳定地结合到单链DNA上（中井，H.和理查森，C.C.（1986年）《生物化学杂志》261，15208 - 15216）。基因4蛋白 - T7 DNA聚合酶 - 模板复合物可在核苷5'-三磷酸存在或不存在的情况下形成。通过凝胶过滤可以分离出不含未结合蛋白质和核苷酸的蛋白质 - 模板复合物，并且该复合物在ATP、CTP和四种脱氧核苷5'-三磷酸存在的情况下能催化以RNA为引物的DNA合成。以RNA为引物的DNA合成可分为引物合成和DNA合成的单独反应。将基因4蛋白与单链DNA、ATP和CTP一起温育时，会形成引物 - 模板复合物；很可能基因4蛋白介导寡核苷酸与模板的稳定结合。该复合物经过纯化，不含未结合的蛋白质和核苷酸，在加入DNA聚合酶和脱氧核苷5'-三磷酸后可支持DNA合成。在引物合成过程中，dTTP或DNA聚合酶的存在会增加引物与模板的结合。引物 - 模板复合物支持的DNA合成主要在基因4识别序列处起始，这表明引物在这些位点与模板结合。
