Mendelman L V, Beauchamp B B, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, MA 02115.
EMBO J. 1994 Aug 15;13(16):3909-16. doi: 10.1002/j.1460-2075.1994.tb06702.x.
Gene 4 of bacteriophage T7 encodes two proteins, a 63 kDa and a colinear 56 kDa protein. The coding sequence of the 56 kDa protein begins at the residues encoding an internal methionine located 64 amino acids from the N-terminus of the 63 kDa protein. The 56 kDa gene 4 protein is a helicase and the 63 kDa gene 4 protein is a helicase and a primase. The unique 7 kDa N-terminus of the 63 kDa gene 4 protein is essential for primer synthesis and contains sequences with homology to a Cys4 metal binding motif, Cys-X2-Cys-X17-Cys-X2-Cys. The zinc content of the 63 kDa gene 4 protein is 1.1 g-atom/mol protein, while the zinc content of the 56 kDa gene 4 protein is < 0.01, as determined by atomic absorption spectrometry. A bacteriophage deleted for gene 4, T7 delta 4-1, is incapable of growing on Escherichia coli strains that contain plasmids expressing gene 4 proteins with single amino acid substitutions of Ser at each of the four conserved Cys residues (efficiency of plating, 10(-7)). Primase containing a substitution of the third Cys for Ser has been overexpressed in E. coli and purified to homogeneity. This mutant primase cannot catalyze template-directed synthesis of oligoribonucleotides although it is able to catalyze the synthesis of random diribonucleotides in a template-independent fashion. The mutant primase has reduced helicase activity although it catalyzes single-stranded DNA-dependent hydrolysis of dTTP at rates comparable with wild type primase. The zinc content of the mutant primase is 0.5 g-atom/mol protein.
T7噬菌体的基因4编码两种蛋白质，一种是63 kDa的蛋白质，另一种是与其共线性的56 kDa蛋白质。56 kDa蛋白质的编码序列始于编码一个内部甲硫氨酸的残基，该甲硫氨酸位于63 kDa蛋白质N端64个氨基酸处。56 kDa的基因4蛋白质是一种解旋酶，而63 kDa的基因4蛋白质既是解旋酶又是引发酶。63 kDa基因4蛋白质独特的7 kDa N端对于引物合成至关重要，并且包含与Cys4金属结合基序Cys-X2-Cys-X17-Cys-X2-Cys具有同源性的序列。通过原子吸收光谱法测定，63 kDa基因4蛋白质的锌含量为1.1克原子/摩尔蛋白质，而56 kDa基因4蛋白质的锌含量<0.01。缺失基因4的噬菌体T7 delta 4-1无法在含有表达基因4蛋白质的质粒的大肠杆菌菌株上生长，这些基因4蛋白质在四个保守的Cys残基处的每一个都被单个氨基酸Ser取代（平板接种效率为10^(-7)）。含有第三个Cys被Ser取代的引发酶已在大肠杆菌中过表达并纯化至同质。这种突变引发酶虽然能够以模板非依赖的方式催化随机二核苷酸的合成，但不能催化模板指导的寡核糖核苷酸的合成。该突变引发酶的解旋酶活性降低，尽管它催化dTTP的单链DNA依赖性水解的速率与野生型引发酶相当。突变引发酶的锌含量为0.5克原子/摩尔蛋白质。
