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.
Mendelman
L V
LV
Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, MA 02115.
Beauchamp
B B
BB
Richardson
C C
CC
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
D004277:DNA, Single-Stranded; D008667:Metalloproteins; D009843:Oligoribonucleotides; D013942:Thymine Nucleotides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases; D015032:Zinc; C024157:thymidine 5'-triphosphate