title: Vaccinia virus mRNA (guanine-7-)methyltransferase: mutational effects on cap methylation and AdoHcy-dependent photo-cross-linking of the cap to the methyl acceptor site.
abstract: The (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme is composed of the C-terminal portion of the D1 subunit, D1(498-844), heterodimerized with the D12 protein. In order to identify protein structural elements involved in cap methylation, we introduced eight alanine substitution mutations within two sequence motifs of D1(498-844)-(594)VLAIDFGNG(602) and (681)IHYSF(685)--that are conserved in the cap methyltransferase from yeast. The D1(498-844)-Ala proteins were coexpressed in bacteria with the D12 subunit, and the recombinant D1(498-844)/D12 heterodimers were purified. Alanine substitutions at five positions--Asp-598, Gly-602, Ile-681, Ser-684, and Phe-685--had little or no effect on methyltransferase activity. Mutations at three conserved residues were deleterious. Alanine substitution at Gly-600 reduced the specific activity to 4% of that of the wild-type 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 established that the aromatic group was essential for cap methylation, whereas the hydroxyl moiety was dispensable. Specific binding of the methyltransferase to the RNA cap was demonstrated by UV cross-linking to [32P]GMP-labeled capped poly(A). Label transfer occurred exclusively to the D1(498-844) subunit and was competed by the cap analogs GpppA and m7GpppA. Cap-specific cross-linking to m7GpppA(pA)n was stimulated by AdoHcy, whereas cross-linking to GpppA(pA)n was unaffected by AdoHcy, but 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, Y683A, and Y683S mutant methyltransferases were unable to cross-link to the cap in the presence of AdoHcy. The catalytically defective G600A mutant did cross-link to the cap in the presence of AdoHcy, suggesting that this mutation affects the chemical step of transmethylation.
authors.affiliation: Molecular Biology Program, Sloan Kettering Institute, New York, New York 10021, USA.
authors.forename: X
authors.initials: X
authors.lastname: Mao
authors.forename: S
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D004926:Escherichia coli; D006150:Guanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012315:RNA Caps / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity; D014466:Ultraviolet Rays; D014616:Vaccinia virus; D000098556:mRNA Guanylyltransferases
chemical_list: D003432:Cross-Linking Reagents; D006150:Guanine Nucleotides; D012315:RNA Caps; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D008274:Magnesium; D000409:Alanine