Location of the S-adenosyl-L-methionine binding region of the vaccinia virus mRNA (guanine-7-)methyltransferase.
The mRNA (guanine-7-)methyltransferase activity of the heterodimeric vaccinia virus mRNA capping enzyme was previously mapped to the carboxyl-terminal 396 amino acids of the large subunit, D1R. This activity is enhanced 30- to 50-fold by the association of the the small subunit, D12L (Higman, M. A., Christen, L. A., and Niles, E. G. (1994) J. Biol. Chem. 269, 14974-14981). Irradiation with ultraviolet light specifically photolinks S-[methyl-3H]S-adenosyl-L-methionine to the large subunit of both the intact capping enzyme and a methyltransferase subdomain, which consists of the D12L subunit associated with the carboxyl-terminal 346 amino acids of the D1R subunit. The extent of linkage was shown to be dependent on the length of incubation, intensity of ultraviolet light, and the concentration of both active enzyme and substrate. The covalent modification was inhibited by S-adenosyl-L-homocysteine, a known competitive inhibitor of the mRNA (guanine-7-)methyltransferase activity, demonstrating specific photolabeling of the active site. Sequence-specific chemical cleavage of the photolinked methyltransferase domain with mild acid or cyanogen bromide revealed linkage of S-adenosyl-L-methionine to two regions of the large subunit. Analysis of the products of cyanogen bromide and hydroxylamine cleavage mapped the photolinked fragments to amino acids 499 to 579 and 806 to 844 of the D1R subunit. Photolinkage of AdoMet to D1R498-844 was shown to be unaffected by the association of the small subunit.
Higman
M A
MA
Department of Biochemistry, State University of New York, School of Medicine and Biomedical Sciences, Buffalo 14214.
Niles
E G
EG
D001665:Binding Sites; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D012436:S-Adenosylmethionine / Q000378:metabolism*; D014466:Ultraviolet Rays; D014616:Vaccinia virus / Q000201:enzymology*; D014764:Viral Proteins
D009097:Multienzyme Complexes; D014764:Viral Proteins; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus