E COLI PRIMASE ZINC STRUCTURE IS SENSITIVE TO BINDING OF ATP & HIGH MAGNESIUM

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The structure of the single zinc site in primase from Escherichia coli was studied using X-ray absorption spectroscopy (XAS). The zinc site in native primase was found to be tetrahedrally ligated by three sulfurs at an average distance of 2.37 ?0.02E and one histidine nitrogen located at a distance of 2.11? 0.02 E. Addition of high concentrations of Mg ?or Mn?- acetate causes an increase in the coordination of the zinc by one or two N/O ligands so that the average N/O ligand distance is 2.19 1 0.03 E. When ssDNA was added to primase, Hthe zinc site structure also increases the coordination by one or two HN/O ligands, but the average distance is 2.05 ? 0.03 E, significantly Hdifferent from the addition of ATP which gave results within the error Hof Mg ?or Mn?-acetate. The higher shells show considerable changes Hand support a conformation change. Thus, the zinc site is altered to Hoctahedral coordination by three sulfur and three oxygen (or nitrogen) Hligands, one of which could be a histidine nitrogen. These results Hand others indicate that the primase zinc site is similar to the Hessential zinc site from Escherichia coli RNA polymerase which can Halso be coordinated by its initiating nucleotide ATP (Wu, F.Y.H., HHuang, W.J., Sinclair, R.B., Powers, L. Journal of Biological HChemistry 267: 25560-25567, 1992. In light of this, the high-magnesium conformation result suggests that high magnesium primase becomes inactive because the zinc is prevented from coordinating ATP.