Interaction and mutual stabilization of the two subunits of vaccinia virus mRNA capping enzyme coexpressed in Escherichia coli.
The genes D1 and D2, predicted to encode the 95- and 31-kDa subunits of the vaccinia virus mRNA capping enzyme, were coexpressed from the same plasmid in Escherichia coli. Induction with low concentrations of isopropyl beta-D-thiogalactoside was necessary to obtain soluble enzyme. The active heterodimer was purified by column chromatography and was shown to have both RNA guanylyltransferase and mRNA (guanine-N7-)-methyltransferase activities. Formation of the m7G(5')pppG cap structure was verified by enzyme digestion and thin-layer chromatography. Each subunit was also expressed individually in E. coli. Without the large subunit, the small one was very unstable in some bacterial strains and could only be detected by pulse labeling with radioactive amino acids. The individually expressed large subunit contained the guanylyltransferase domain, but the activity from E. coli was less than 2% of that obtained with both subunits. Two other products of the D1 open reading frame were formed: a 55-kDa subfragment with the GMP binding site and a 38-kDa C-terminal fragment that started at amino acid 498. Expression of this heterodimeric enzyme in E. coli may facilitate the analysis of its functional domains and provide a useful reagent for the specific 5' labeling of uncapped or capped RNA and for enhancing RNA translatability in eukaryotic systems.
Guo
P X
PX
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Moss
B
B
D003001:Cloning, Molecular; D004926:Escherichia coli; D005814:Genes, Viral; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics* / Q000302:isolation & purification; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000302:isolation & purification; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics* / Q000302:isolation & purification; D012315:RNA Caps; D011994:Recombinant Proteins / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics
D009097:Multienzyme Complexes; D012315:RNA Caps; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus