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

The Journal of Biological Chemistry
A A Mustaev, G N Godson

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.

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Citations

Jun 26, 1997·Biochimica Et Biophysica Acta·P SzafranskiC R Cantor
Jul 16, 2010·Nature Reviews. Molecular Cell Biology·Malin AkerfeltLea Sistonen
Jan 31, 2002·Proceedings of the National Academy of Sciences of the United States of America·Tianxin Chen, Carl S Parker
Jul 12, 2003·Journal of Biomolecular Structure & Dynamics·Dmitry M Kolpashchikov
Sep 14, 2002·Proceedings of the National Academy of Sciences of the United States of America·Seung-Joo Lee, Charles C Richardson
Mar 23, 2011·Annual Review of Biochemistry·Julius Anckar, Lea Sistonen
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