Enriched sources of Escherichia coli replication proteins. The dnaG primase is a zinc metalloprotein.
Primase, the product of the Escherichia coli dnaG gene, is the enzyme responsible for RNA primer synthesis on both template strands at replication forks during chromosomal DNA synthesis. The dnaG gene was modified by replacement of the natural ribosome-binding site with one complementary to the 3' end of 16S rRNA, and then inserted downstream of tandem bacteriophage lambda PR and PL promoters in the pUC9-derived vector pCE30. Following thermal induction of transcription, the resulting plasmid pPL195 directed synthesis of primase activity to levels corresponding to approx. 120,000 molecules per cell. The overproduced protein was soluble and was readily purified in high yield (31 mg per 1 of culture). Purified primase was monomeric, was fully active in priming replication at the bacteriophage G4 complementary strand origin, and was shown to contain 0.92 +/- 0.08 g atom of tightly-bound zinc per mol of protein. Potential zinc-binding amino-acid residues near the N-terminus of the protein were identified. Although a mutant primase lacking 27 amino acid residues from the N-terminus was partly soluble, it was completely inactive.
Stamford
N P
NP
Centre for Molecular Structure and Function, Research School of Chemistry, Australian National University, Canberra, ACT.
Lilley
P E
PE
Dixon
N E
NE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D019915:DNA Primase; D004261:DNA Replication / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008667:Metalloproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012316:RNA Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D016335:Zinc Fingers / Q000235:genetics*
D008667:Metalloproteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases