Magnesium Acetate Induces a Conformational Change in Escherichia coli Primase

Primase from Escherichia coli is a single-stranded DNA-dependent RNA polymerase. As such, it requires magnesium to carry out catalysis. Limited tryptic digestion was used to probe the conformations of primase as a function of magnesium acetate concentration. In the absence of magnesium, trypsin cleaved primase at three sites. Magnesium acetate induced a conformational change such that one of these sites became inaccessible to trypsin digestion and a new site became trypsin accessible. The conformational change was only induced by Mg(OAc)₂ and not MnCh, CaCL, NaOAc or LiCl, indicating a clear magnesium acetate-dependent conformational change. The effect was slightly induced by MgSO₄ and MgCh. An allosteric binding model indicates that primase binds at least two magnesiums in a cooperative manner. The data were best fit to a two-state model in which one conformation had a high affinity for magnesium, K_R = 83.4 M^-1, and the other state had virtually no affinity.