Studies of the functional topography of the catalytic center of Escherichia coli primase

Abstract

The catalytic center of E. coli primase (581 amino acids) was identified by using, in the G4ori(c) single-strand binding protein (SSB) primer RNA (pRNA) synthesis system, ATP and AMP derivatives, which were modified on the 5' side with reactive groups that can be cross-linked to the ATP binding site plus [α-32P]GTP. The position of the covalently attached 32P-labeled dinucleotide was mapped by chemical and enzymatic cleavage of labeled wild type and deletion mutants of primase. The catalytic center involves one of the Lys residues Lys-211, Lys-229, and Lys-241. The ATP binding site is preformed in primase, and the cross-linked ATP residue can be elongated to a 5-nucleotide limit, which implies significant stretching of the catalytic center during pRNA synthesis. His-43 close to the N terminus in a proposed zinc finger and Lys-528 near the C terminus were also cross-linked to ATP residues in the primase ATP binding site, suggesting that these regions are topographically close to the catalytic center during pRNA synthesis. When cross-linking was performed on the preformed primase/SSB/G4ori(c) complex with long arm reagents (12-15 Å), SSB was also labeled, indicating a close proximity to the site of pRNA synthesis.