Figure 6.

Kinetic profile of guanylate transfer to an RNA 3′-phosphate. (A) RtcB was reacted with pRNAp or _HORNAp in the presence of 0.1 mM GTP as described in Figure 5. The reactions were quenched with EDTA at the times specified. (A control mixture containing RtcB and pRNAp but no added GTP was incubated for 120 s). The RNAs were digested with RNase T1, and the products were analyzed by Urea–PAGE and visualized by autoradiography. (B) The extent of conversion of the pRNAp substrate to pRNAppG, expressed as the percent of the total radiolabeled material, is plotted as a function of reaction time. Each datum is the average of four independent experiments ± SEM. (Three of the experiments were performed without RNase T1 treatment as in Figure 5 and one experiment was performed with RNase T1 treatment as in panel A).