The mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme.
The mRNA (guanine-7-)methyltransferase active site of the heterodimeric vaccinia virus mRNA capping enzyme was previously localized to the carboxyl-terminal third of the large subunit, D1R, associated with the small subunit, D12L (Cong, P., and Shuman, S. (1992) J. Biol. Chem. 267, 16424-16429; Higman, M. A., Bourgeois, N., and Niles, E. G. (1992) J. Biol. Chem. 267, 16430-16437). A plasmid was constructed which directs the coexpression of the carboxyl terminus of the D1R subunit from amino acids 498 to 844 and the D12L subunit in Escherichia coli. The mRNA (guanine-7-)methyltransferase catalytic activity in the isolated domain was found to be kinetically equivalent to that present in the intact enzyme. Through mobility shift and ultraviolet photolinkage analyses, both domains were shown to bind RNA in a saturable fashion. RNA binding was localized predominantly to the large subunit, but a low level of linkage of RNA to D12L was also observed. A low, but reproducible, level of mRNA (guanine-7-)methyltransferase activity was detected in the isolated D1R498-844 subunit demonstrating that the active site resides solely within the large subunit of the capping enzyme. This activity is enhanced 30- to 50-fold by the association of the D12L subunit.
Higman
M A
MA
Department of Biochemistry, State University of New York, School of Medicine and Biomedical Sciences, Buffalo 14214.
Christen
L A
LA
Niles
E G
EG
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010777:Photochemistry; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins
D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014764:Viral Proteins; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus