In order to characterize the inhibition of the GTase activity by MPA, we next set out to investigate which of the two catalytic steps of the reaction is inhibited by MPA. The first step of the reaction, i.e. the formation of the enzyme-GMP covalent intermediate, was monitored by incubating the purified enzyme in the presence of [alpha-32 P]GTP. The radiolabeled covalent enzyme-GMP complex was then visualized by autoradiography following electrophoresis on a denaturant polyacrylamide gel. Our results indicate that very high concentrations of MPA are required to inhibit the formation of the enzyme-GMP covalent complex (Fig. 2C) . We determined that a concentration of 3 mM of MPA is required to inhibit the first step of the reaction by 50%, a concentration that corresponds to 8-times the amount required to inhibit the overall GTase reaction (both steps). The effect of MPA on the second step of the RNA reaction (i.e. the transfer of the GMP moiety onto an acceptor RNA) was next evaluated by pre-incubating the enzyme with GTP to ensure formation of the covalent enzyme-GMP complex, followed by the addition of the acceptor 59-diphosphate RNA in the presence of MPA. Using such an approach, it was determined that a concentration of 640 mM of MPA is sufficient to inhibit 50% of the second step of the GTase reaction (Fig. 2D) . Taken together, these results indicate that MPA inhibits the GTase reaction mainly through inhibition of the catalytic transfer of the GMP moiety onto an acceptor RNA.