ATP Cross-Linked to Escherichia coli Single-Strand DNA-Binding Protein Can Be Utilized by the Catalytic Center of Primase as Initiating Nucleotide for Primer RNA Synthesis on Phage G4oric Template

• Published in: Biochemistry (Easton) 1998-03, Vol.37 (11), p.3810-3817
• Main Authors: Godson, G. Nigel, Mustaev, A. Arkady, Sun, Wuliang
• Format: Article
• Language: English
• Subjects: Adenine Nucleotides - metabolism; Adenosine Triphosphate - metabolism; Bacteriophage T7 - genetics; Catalysis; Cross-Linking Reagents - metabolism; DNA Primase - metabolism; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - metabolism; Escherichia coli - genetics; Oligoribonucleotides - metabolism; Organophosphates - metabolism; Recombinant Fusion Proteins - metabolism; Replication Origin - genetics; RNA - biosynthesis; RNA - metabolism; Templates, Genetic
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi972455f

We report a new observation of the role of Escherichia coli single-strand DNA binding protein (SSB) in synthesis of primer RNA (pRNA) catalyzed by E. coli primase on the SSB-coated phage G4oric template. Using a set of ATP priming substrates with reactive groups attached to the 5‘ γ-phosphate on different length “arms”, we have demonstrated that, in the primase/SSB/G4oric pRNA synthesis complex, ATP cross-linked to both primase and SSB could be equally utilized as initiating nucleotide for pRNA synthesis. The distance between SSB surface and α-phosphorus of the priming substrate was estimated to be less than 7 Å. ATP cross-linked to primase and SSB can be further elongated in the presence of other NTPs, giving almost identical patterns of covalently attached pRNAs of up to 12 nucleotides in length. The regions of primase and SSB with cross-linked ATP that can be used for pRNA synthesis are, therefore, arranged in a similar way relative to the active center of pRNA synthesis. The pRNA covalently linked to SSB was localized, mapping between Met48 and Trp88. This observation raises the possibility that SSB may play an active role in the initiation of pRNA synthesis in this system.