Domain structure of vaccinia virus mRNA capping enzyme. Activity of the Mr 95,000 subunit expressed in Escherichia coli.
The D1 gene encoding the large subunit of vaccinia virus mRNA capping enzyme was expressed in Escherichia coli BL21(DE3) under the control of a bacteriophage T7 promoter. Guanylyltransferase activity (assayed as the formation of a covalent enzyme-guanylate complex) was detected in soluble lysates of these bacteria. Two major species of protein-GMP complex were formed, one of Mr 95,000 (corresponding in size to the D1 gene product) and one of Mr 60,000. Partial purification of the guanylyltransferase was effected by ammonium sulfate precipitation and ion-exchange chromatography. The expressed large subunit synthesized GpppA caps when provided with 5'-triphosphate-terminated poly(A) as a cap acceptor, but was unable to catalyze cap methylation in the presence of S-adenosylmethionine. Thus, the small capping enzyme subunit was shown to be dispensable for guanylylation, but required for cap methylation of RNA. The Mr 95,000 and Mr 60,000 protein-GMP forming activities were resolved during centrifugation in a glycerol gradient; the two forms sedimented at 5.5 S and 4.4 S, respectively, consistent with each enzyme form being a monomer. Either species catalyzed GMP transfer to an RNA acceptor. The isolated Mr 95,000 guanylyltransferase could be converted to an active Mr 60,000 form in vitro by limited proteolysis with trypsin. Expression of carboxyl-deleted forms of the D1 gene product in E. of carboxyl-deleted forms of the D1 gene product in E. coli further localized the guanylyltransferase domain to the amino two-thirds of the Mr 95,000 polypeptide.
Shuman
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Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021.
Morham
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D002499:Centrifugation, Density Gradient; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005814:Genes, Viral; D046911:Macromolecular Substances; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics; D000098556:mRNA Guanylyltransferases
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus