Domain structure of the vaccinia virus mRNA capping enzyme: expression in Escherichia Coli of a subdomain possessing the RNA 5′-triphosphatase and guanylyltransferase activities and a kinetic comparison to the full-size enzyme

The RNA 5′-triphosphatase, nucleoside triphosphate phosphohydrolase, and guanylyltransferase activities of the vaccinia virus mRNA capping enzyme were previously localized to an NH₂-terminal 60-kDa domain of the DIR subunit. Measurement of the relative ATPase and guanylyltransferase activities remaining in DIR carboxyl-terminal deletion variants expressed in Escherichia coll BL21(DE3)plysS localizes the carboxyl terminus of the active domain to between amino acids 520 and 545. Failure to obtain a deletion mutant with the loss of one activity indicates that the catalysis of both reactions requires a common domain structure. Based on these results, a truncated DIR protein terminating at amino acid 545 was expressed in E. coll and purified to homogeneity. DIR^1-545 was found to be kinetically equivalent to the holoenzyme in regard to ATPase, RNA 5′-triphosphatase, and guanylyltransferase activities. Measurement of RNA binding by mobility shift and UV photocross-linking analyses also demonstrates the ability of this domain to bind RNA independent of the methyltransferase domain, comprised of the carboxyl terminus of DIR from amino acids 498-844 and the entire D12L subunit. RNA binding to DIR^1-545 is substantially weaker than binding to either the methyltransferase domain or the holoenzyme. Binding is inhibited by 5′-OH RNA and to a lesser extent by DNA oligonucleotides in a concentration dependent manner which correlates with the inhibition of RNA 5′-triphosphatase activity by these same oligonucleotides. We conclude that DIR^1-545 represents a functionally independent domain of the mRNA capping enzyme, fully competent in substrate binding and catalysis at both the triphosphatase and guanylyltransferase active sites.