RNA triphosphatase,
RNA guanylyltransferase, and
RNA (guanine-7)-methyltransferase activities are associated with the vaccinia virus
mRNA capping enzyme, a heterodimeric
protein containing
polypeptides of Mr 95,000 and Mr 31,000. The genes encoding the large and small subunits (corresponding to the D1 and the D12 ORFs, respectively, of the viral genome) were coexpressed 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. A 1000-fold purification of the
guanylyltransferase was achieved by
ammonium sulfate precipitation and chromatography using
phosphocellulose and SP5PW columns. Partially purified guanylytransferase synthesized GpppA caps when provided with 5'-triphosphate-terminated
poly(A) as a cap acceptor. In the presence of
AdoMet the
enzyme catalyzed concomitant cap methylation with 99% efficiency. Inclusion of S-adenosyl
methionine increased both the rate and extent of
RNA capping, permitting quantitative modification of
RNA 5' ends.
Guanylyltransferase sedimented as a single component of 6.5 S during further purification in a
glycerol gradient; this S value is identical with that of the heterodimeric capping
enzyme from
vaccinia virions. Electrophoretic analysis showed a major
polypeptide of Mr 95,000 cosedimenting with the
guanylyltransferase.
RNA triphosphatase activity cosedimented exactly with
guanylyltransferase.
Methyltransferase activity was associated with
guanylyltransferase and was also present in less rapidly sedimenting fractions. The
methyltransferase activity profile correlated with the presence of a Mr 31,000
polypeptide. These results indicate that the D1 and D12 gene products are together sufficient to catalyze all three enzymatic steps in cap synthesis. A model for the domain structure of this
enzyme is proposed.