VACCINIA VIRUS MESSENGER-RNA (GUANINE-7-)METHYLTRANSFERASE - MUTATIONAL EFFECTS ON CAP METHYLATION AND ADOHCY-DEPENDENT PHOTO-CROSS-LINKINGOF THE CAP TO THE METHYL ACCEPTOR SITE

The (guanine-7-) methyltransferase domain of-the vaccinia virus mRNA c apping enzyme is composed of the C-terminal portion of the D1 subunit, D1(498-844), heterodimerized with the D12 protein, In order to identi fy protein structural elements involved in cap methylation, we introdu ced eight alanine substitution mutations within two sequence motifs of D1(498 - 844)-(594) VLAIDFGNG(602) and (681)IHYSF(685)-that are conse rved in the cap methyltransferase from yeast. The D1(498-844)-Ala prot eins were coexpressed in bacteria with the D12 subunit, and the recomb inant D1(498-844)/D12 heterodimers were purified. Alanine substitution s at five positions-Asp-598, Gly-602, Ile-681, Ser-684, and Phe-685-ha d little or no effect on methyltransferase activity. Mutations at thre e conserved residues were deleterious. Alanine substitution at Gly-600 reduced the specific activity to 4% of that of the wildtype protein, Substitutions at His-682 and Tyr-683 reduced activity to 4% and 0.05%, respectively. By further mutating Tyr-683 to Phe and Ser, we establis hed that the aromatic group was essential for cap methylation, whereas the hydroxyl moiety was dispensable. Specific binding of the methyltr ansferase to the RNA cap was demonstrated by UV cross-linking to [P-32 ]GMP-labeled capped poly(A). Label transfer occurred exclusively to th e D1(498-844) subunit and was competed by the cap analogs GpppA and m7 GpppA. Cap-specific cross-linking to m7GpppA(pA)n was stimulated by Ad oHcy, whereas cross-linking to GpppA(pA)n was unaffected by AdoHcy, bu t stimulated by AdoMet. We suggest that occupancy of the methyl donor site either enhances the affinity for the cap guanosine or alters the protein interface so that a photoreactive moiety is brought closer to the cap structure. The catalytically defective H682A, YS83A, and Y683S mutant methyltransferases were unable to cross-link to the cap in the presence of AdoHcy. The catalytically defective G600A mutant did cros s-link to the cap in the presence of AdoHcy, suggesting that this muta tion affects the chemical step of transmethylation.