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 collin ear 56 kDa protein. The coding sequence of the 56 kDa protein begins a t 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 i s 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 essentia l for primer synthesis and contains sequences with homology to a Cys(4 ) metal binding motif, Cys-X(2)-Cys-X(17)-Cys-X(2)-Cys. The zinc conte nt of the 63 kDa gene 4 protein is 1.1 g-atom/mol protein, while the z inc content of the 56 kDa gene 4 protein is <0.01, as determined by at omic absorption spectrometry. A bacteriophage deleted for gene 4, T7 D elta 4-1, is incapable of growing on Escherichia coli strains that con tain plasmids expressing gene 4 proteins with single amino acid substi tutions of Ser at each of the four conserved Cys residues (efficiency of plating, 10(-7)). Primase containing a substitution of the third Cy s for Ser has been overexpressed in E.coli and purified to homogeneity . This mutant primase cannot catalyze template-directed synthesis of o ligoribonucleotides although it is able to catalyze the synthesis of r andom diribonucleotides in a template-independent fashion. The mutant primase has reduced helicase activity although it catalyzes single-str anded DNA-dependent hydrolysis of dTTP at rates comparable with wild t ype primase. The zinc content of the mutant primase is 0.5 g-atom/ mol protein.