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

We report a new observation of the role of Escherichia coli single-str and DNA binding protein (SSB) in synthesis of primer RNA (pRNA) cataly zed 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 ' gamma-phosphate on different length ''arms'', we have demonstrated t hat, in the primase/SSB/G4oric pRNA synthesis complex, ATP cross-linke d to both primase and SSB could be equally utilized as initiating nucl eotide for pRNA synthesis. The distance between SSB surface and ex-pho sphorus of the priming substrate was estimated to be less than 7 Angst rom. ATP cross-linked to primase and SSB can be further elongated in t he presence of other NTPs, giving almost identical patterns of covalen tly attached pRNAs of up to 12 nucleotides in length. The regions of p rimase and SSB with cross Linked ATP that can be used for pRNA synthes is are, therefore, arranged in a similar way relative to the active ce nter of pRNA synthesis, The pRNA covalently linked to SSB was localize d, mapping between Met48 and Trp88. This observation raises the possib ility that SSB may play an active role in the initiation of pRNA synth esis in this system.