| Abstract: |
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 M(r) 95,000 (corresponding in size to the D1 gene product) and one of M(r) 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 M(r) 95,000 and M(r) 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 M(r) 95,000 guanylyltransferase could be converted to an active M(r) 60,000 form in vitro by limited proteolysis with trypsin. Expression of carboxyl-deleted forms of the D1 gene product in E. coli further localized the guanylyltransferase domain to the amino two-thirds of the M(r) 905,000 polypeptide. |
| Keywords: |
unclassified drug; genetics; mutation; nonhuman; metabolism; gene expression; phosphatase; enzymology; molecular cloning; cloning, molecular; methyltransferase; methyltransferases; escherichia coli; recombinant proteins; recombinant protein; vaccinia virus; multienzyme complex; multienzyme complexes; chromosome deletion; isolation and purification; molecular weight; phosphoric monoester hydrolases; centrifugation, density gradient; macromolecule; nucleotidyltransferase; vaccinia; rna capping; nucleotidyltransferases; structural gene; guanylyltransferase; density gradient centrifugation; messenger ribonucleic acid guanylyltransferase; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; macromolecular systems; capping enzyme, vaccinia virus; genes, structural, viral
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