Abstract
The catalytic center of E. coli primase (581 amino acids) was identified by using, in the G4oric 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 [alpha-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/G4oric complex with long arm reagents (12-15 A), SSB was also labeled, indicating a close proximity to the site of pRNA synthesis.
Highlights
From the Public Health Research Institute, New York, New York 10016 and the t.Biochemistry Department, New York University Medical Center, New York, New York 10016
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 primer RNA (pRNA) synthesis
Both the C-terminal and N-terminal regions of primase appear to be close to the catalytic center
Summary
Vol 270, No 26, Issue of June 30, pp. 15711-15718, 1995 Printed in U.S.A. Studies of the Functional Topography of the Catalytic Center of Escherichia coli Primase*. The catalytic center of E. coli primase (581 amino acids) was identified by using, in the G4oric 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 crosslinked to the ATP binding site plus [a-32P]GTP. A sequence containing part of these two motifs is conserved between primases and the large subunit of DNA-dependent RNA polymerases [16] It has been demonstrated using proteolytic cleavage that primase consists of two physical domains: a large 47-kDa N-terminal domain that retains the pRNA synthesis activity and a 16-kDa Cterminal domain that appears to be involved in the interaction of primase with its accessory proteins [17, 18]. A His amino acid residue that is part of the postulated N-terminal zinc finger was cross-linked to the active center
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