Figure 2. Mycophenolic acid inhibits the RNA guanylyltransferase activity.

(A) Molecular structure of mycophenolic acid (MPA). (B) Increasing concentrations of MPA inhibit the complete RNA guanylyltransferase reaction. A standard GTase assay in which the purified enzyme (1 µM) was incubated with both [alpha-³²P]GTP and a 5′-diphosphate acceptor RNA was performed in the presence of increasing concentrations of MPA. The reaction products were analyzed on a denaturing polyacrylamide gel and quantified (right side of the panel). (C) MPA is not a potent inhibitor of the first step of the GTase reaction. The formation of the enzyme-GMP covalent intermediate was monitored by incubating the purified enzyme (1 µM) in the presence of [alpha-³²P]GTP and increasing concentrations of MPA. The radiolabeled covalent enzyme-GMP complex was then visualized by autoradiography following electrophoresis on a denaturing 12.5% polyacrylamide gel. The radiolabeled enzyme-GMP complex was quantified by phosphorimaging (right side of the panel). (D) The second step of the GTase reaction is inhibited by MPA. The transfer of the GMP moiety onto an acceptor RNA was evaluated by pre-incubating the enzyme (1 µM) with [alpha-³²P]GTP (10 mM) to ensure formation of the radiolabeled covalent enzyme-GMP complex, followed by the addition of the acceptor 5′-diphosphate RNA (3 µM) in the presence of MPA. Formation of the radiolabeled capped GpppRNA was monitored following electrophoresis on a denaturant polyacrylamide gel. The radiolabeled GpppRNA was then quantified by phosphorimaging (right side of the panel).