Functional domains of vaccinia virus mRNA capping enzyme. Analysis by limited tryptic digestion.
RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7)-methyltransferase, and transcription termination factor activities are associated with the mRNA capping enzyme from vaccinia virus. Purified vaccinia capping enzyme is a 6.5 S protein containing two subunits of Mr = 95,000 and Mr = 31,000. Although the RNA guanylyltransferase domain has been localized to the large subunit by virtue of the formation of a Mr = 95,000 covalent protein-GMP intermediate, the location of other functional domains within the protein and the catalytic role of individual subunits remain unclear. In the present study, limited proteolysis with trypsin was shown to convert the vaccinia capping enzyme into a form capable of generating a Mr = 59,000 enzyme-GMP complex. Purification of the trypsinized enzyme by glycerol gradient sedimentation resulted in the isolation of a 4.2 S fragment of the large subunit that retains RNA triphosphatase and RNA guanylyltransferase activities. This derivative, containing little or no small subunit (or fragments thereof), has lost the ability to catalyze methyl group transfer and to mediate transcription termination in vitro. Residual methyltransferase activity was found associated with a minor 5.2 S tryptic product that cosediments with a Mr = 21,000 fragment of the small enzyme subunit. A model for the organization of functional domains within the capping enzyme is suggested.
Shuman
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Program in Molecular Biology, Sloan-Kettering Institute, New York 10021.
D006868:Hydrolysis; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D010454:Peptide Termination Factors / Q000032:analysis / Q000502:physiology; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D012315:RNA Caps / Q000032:analysis*; D012333:RNA, Messenger / Q000378:metabolism*; D014357:Trypsin; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*
D009097:Multienzyme Complexes; D010454:Peptide Termination Factors; D012315:RNA Caps; D012333:RNA, Messenger; D014764:Viral Proteins; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus; D014357:Trypsin