FUNCTIONAL DOMAINS OF VACCINIA VIRUS MESSENGER-RNA CAPPING ENZYME - ANALYSIS BY LIMITED TRYPTIC DIGESTION

  • 5 June 1989
    • journal article
    • research article
    • Vol. 264  (16) , 9690-9695
Abstract
RNA triphosphatase, RNA guanylytransferase, RNA (guanine-7)-methyltransferase, and transcription termination factor activities are associated with the mRNA cappping enzyme form 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 guanylytransferase domain has been localized to the large subunit by virtue of the formaton 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 isoltion of a 4.2 S fragment of the large subunit that retains RNA triphosphatase and RNA guanylyltransferase activitties. This derivative, containing little or no small subunit (or fragments thereof), has lost the ability to catalyze methyl group transfer and to mediate transcriptioin termination in vitro.Besidual methyltransferase activity was found associted with a minor 5.2 S tryptic product that cosediments with a Mr=21,000 fragmnet of the small enzyme subunit. A model for the organizaiton of functional domains within the capping enzyme is suggested.

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