Intrinsic RNA (guanine-7) methyltransferase activity of the vaccinia virus capping enzyme D1 subunit is stimulated by the D12 subunit. Identification of amino acid residues in the D1 protein required for subunit association and methyl group transfer.
Vaccinia virus mRNA capping enzyme, a heterodimer of virus-encoded D1 and D12 subunits, catalyzes three steps in the synthesis of the m7GpppN cap. By expressing portions of the subunits in bacteria, singly and together, we have localized the RNA (guanine-7) methyltransferase domain to a 305-amino acid carboxyl-terminal segment of the D1 polypeptide (residues 540-844) complexed with the D12 protein. We find that the purified carboxyl D1 protein has a weak intrinsic methyltransferase activity, indicating that the catalytic center resides within this subunit. The basal level of activity can be stimulated 100-fold by addition of purified D12 protein, which is itself catalytically inert. The carboxyl region of D1 forms a heterodimer with the D12 subunit in vivo and in vitro. Analysis of alanine substitution mutants of the D1 protein identifies amino acid residues important for subunit interaction. Our results suggest that subunit heterodimerization is necessary, but not sufficient, for full methyltransferase activity. A mutation of vicinal positions His-682-Tyr-683 that specifically affects catalytic activity but not subunit interaction implicates these residues as constituents of the active site.
Mao
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Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021.
Shuman
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S
D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D008780:Methyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D014616:Vaccinia virus / Q000201:enzymology; D014764:Viral Proteins
D009097:Multienzyme Complexes; D012333:RNA, Messenger; D014764:Viral Proteins; D006639:Histidine; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus