Figure 1.

Analysis of methylated cap structures synthesized by recombinant VP4. Reaction mixtures (100 μl) contain 50 mM Tris⋅HCl (pH7.9), 9 mM MgCl₂, 6 mM DTT, 200 μM GpppG, 2 μMAdo[methyl-³H]Met (87 Ci/mmol), and 15 μg of purified recombinant VP4. Parallel reactions were performed with VP4 protein that had been heated to 68°C for 15 min prior to addition. After incubation for two hr at 30°C and proteinase K treatment, the reaction mix was extracted with phenol/chloroform and precipitated with ethanol. Samples were analyzed by HPLC on a Beckman BioSys 510 system using a SAX 5 ion-exchange column. The products recovered from HPLC were dried and counted in the scintillation counter, and the radioactivity associated with each fraction is plotted. The positions of unincorporated AdoMet (SAM) and m⁷GpppG are indicated. (A) Reaction products from mixtures containing VP4 as enzyme. (B) Reaction products from mixtures containing VP4 that had been heated to 68°C for 15 min prior to addition to the reaction mix. (C) Reanalysis of the peak fraction from A after TAP treatment. The reduction in scale reflects the fact that only ≈10% of the peak from A was analyzed.