title: 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.
abstract: 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 D1R subunit. Measurement of the relative ATPase and guanylyltransferase activities remaining in D1R carboxyl-terminal deletion variants expressed in Escherichia coli 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 D1R protein terminating at amino acid 545 was expressed in E. coli and purified to homogeneity. D1R1-545 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 photo-cross-linking analyses also demonstrates the ability of this domain to bind RNA independent of the methyltransferase domain, comprised of the carboxyl terminus of D1R from amino acids 498-844 and the entire D12L subunit. RNA binding to D1R1-545 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 D1R1-545 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.
authors.affiliation: Department of Biochemistry, State University of New York, School of Medicine and Biomedical Sciences, Buffalo 14214, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Myette
authors.forename: E G
authors.initials: EG
authors.lastname: Niles
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000302:isolation & purification; D012313:RNA / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism*; D014466:Ultraviolet Rays; D014616:Vaccinia virus / Q000201:enzymology*
chemical_list: D012315:RNA Caps; D012313:RNA; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase