Escherichia coli DNA polymerase III holoenzyme forms a stable initiation complex with RNA-primed template in the presence of ATP. To determine the linear arrangement of the holoenzyme subunits along the primer-template duplex region, we cross-linked holoenzyme to a series of photo-reactive primers. Site-specific photo-cross-linking revealed that the , , and subunits formed ATP-dependent contacts with the primer-template. The polymerase catalytic subunit covalently attached to nucleotide positions -3, -9, and -13 upstream of the primer terminus, with the most efficient adduct formation occurring at position -9. The subunit contacted the primer at positions -13, -18, and -22, with the strongest -primer interactions occurring at position -18. The subunit predominated in cross-linking at position -22. Thus, within the initiation complex, contacts roughly the first 13 nucleotides upstream of the 3`-primer terminus followed by at -18 and at -22, and the subunit remains a part of the initiation complex, bridging the and subunits.

Analyses of the interaction of photo-activatible primer-templates with the preinitiation complex proteins (-complex (--`--) and subunit) revealed the subunit within the preinitiation complex covalently attached to primer at position -3. However, addition of core DNA polymerase III to preinitiation complex, fully reconstituting holoenzyme resulted in replacement of by at the primer terminus. These data indicate that assembly of holoenzyme onto a primer-template can occur in distinct stages and results in a structural rearrangement during initiation complex formation.

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