title: 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 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.
authors.affiliation: Public Health Research Institute, New York, New York 10016, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Mustaev
authors.forename: G N
authors.initials: GN
authors.lastname: Godson
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis*; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D004277:DNA, Single-Stranded; D009711:Nucleotides; C034477:RNA primers; D012313:RNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases