FIG. 4.

pRNA synthesis by primase-K241R. A six-His tag was fused to the N terminus of primase by using the vector pET-21d, and HT-primase was prepared by affinity purification on Ni resin. In the same vector, Lys241 was changed to Arg (HT-primase-K241R) by site-directed mutagenesis. (A) Comparison of the activity of HT-primase with two independent preparations of wild-type primase that does not contain a His tag. In this experiment, [α-³²P]UTP was used to label the pRNA products. Care was taken to use identical amounts of primase in each reaction mixture and to load identical aliquots of the reaction mixture on the polyacrylamide gel. (B) pRNA synthesis by HT-primase and HT-primase-K241R was assayed under the standard conditions with [α-³²P]GTP (see Materials and Methods). In both experiments, the synthesis products were separated on a 20% polyacrylamide–7 M urea gel (20 by 40 cm). To visualize the dimer pRNA in panel B, most of the [α-³²P]GTP was run out of the leftmost gel and the bottom region of the frozen gel was autoradiographed for a shorter time, as shown in the rightmost gel.