Requirement for a zinc motif for template recognition by the bacteriophage T7 primase

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.