Methyltransferase and subunit association domains of vaccinia virus mRNA capping enzyme.

RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7-)-methyltransferase activities are associated with the vaccinia virus mRNA capping enzyme, a heterodimeric protein containing polypeptides of M(r) 95,000 and 31,000. Although the RNA triphosphatase and RNA guanylyltransferase domains have been localized to a M(r) 59,000 fragment of the capping enzyme large subunit, the location of the methyltransferase domain within the protein and the catalytic role of individual subunits in methyl group transfer remain unclear. In the present work, through the study of methyltransferase activity of truncated forms of capping enzyme translated in vitro in a rabbit reticulocyte lysate, we have localized the methyltransferase domain to a complex consisting of the small subunit and the carboxyl-terminal portion of the large subunit. The M(r) 31,000 subunit translated alone was not sufficient for methyltransferase activity. This requirement for both subunits may explain the tight physical association of the two polypeptides in vivo. We have recreated the association of the large and small enzyme subunits in vitro through the translation of synthetic mRNAs encoding the two polypeptides. Study of the ability of deleted versions of the large subunit to bind the small subunit, as detected by co-immunoprecipitation, defined a 347-amino acid carboxyl-terminal region of the large subunit that was sufficient for heterodimerization. Colocalization within the large subunit of the methyltransferase and subunit association domains suggests that dimerization of the subunits may be required for methyltransferase activity.     

Catalytic activity of vaccinia mRNA capping enzyme subunits coexpressed in Escherichia coli

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.     

Domain structure of vaccinia virus mRNA capping enzyme. Activity of the Mr 95,000 subunit expressed in Escherichia coli

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 Mr 95,000 (corresponding in size to the D1 gene product) and one of Mr 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 Mr 95,000 and Mr 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 Mr 95,000 guanylyltransferase could be converted to an active Mr 60,000 form in vitro by limited proteolysis with trypsin. Expression of carboxyl-deleted forms of the D1 gene product in E. of carboxyl-deleted forms of the D1 gene product in E. coli further localized the guanylyltransferase domain to the amino two-thirds of the Mr 95,000 polypeptide.     

Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme.

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities. The enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The N-terminal 60-kDa of the D1 subunit (from residues 1 to 545) is an autonomous domain which catalyzes the triphosphatase and guanylyltransferase reactions. Mutations in the D1 subunit that specifically inactivate the guanylyltransferase without affecting the triphosphatase component have been described (P. Cong and S. Shuman, Mol. Cell. Biol. 15:6222-6231, 1995). In the present study, we identified two alanine-cluster mutations of D1(1-545), R77A-K79A and E192A-E194A, that selectively inactivated the triphosphatase, but not the guanylyltransferase. Concordant mutational inactivation of RNA triphosphatase and nucleoside triphosphatase functions (to approximately 1% of wild-type specific activity) suggests that both gamma-phosphate cleavage reactions occur at a single active site. The R77A-K79A and E192A-E194A mutant enzymes were less active than wild-type D1(1-545) in the capping of triphosphate-terminated poly(A) but could be complemented in vitro by D1(1-545)-K260A, which is inert in nucleotidyl transfer but active in gamma-phosphate cleavage. Whereas wild-type D1(1-545) formed only the standard GpppA cap, the R77A-K79A and E192A-E194A enzymes synthesized an additional dinucleotide, GppppA. This finding illuminates a novel property of the vaccinia virus capping enzyme, the use of triphosphate RNA ends as an acceptor for nucleotidyl transfer when gamma-phosphate cleavage is rate limiting.     

Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme)

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.     

RNA capping by HeLa cell RNA guanylyltransferase. Characterization of a covalent protein-guanylate intermediate

RNA capping by partially purified HeLa cell GTP:RNA guanylyltransferase has been shown to occur in the following sequence of two partial reactions involving a covalent protein-guanylate intermediate: (i) E(P68) + GTP in equilibrium E(P68-GMP) + PPi (ii) E(P68-GMP) + ppRNA in equilibrium GpppRNA + E(P68) Initially, the enzyme reacts with GTP in the absence of an RNA cap acceptor to form a covalent protein-guanylate complex. This complex consists of a GMP residue linked via a phosphoamide bond to a Mr = 68,000 protein. The enzyme then transfers the guanylate residue from the Mr = 68,000 polypeptide to the 5' end of diphosphate-terminated poly(a) to yield the capped derivative GpppA(pA)n. Both partial reactions have been shown to be reversible. In the reverse of Reaction i, E(P68--GMP) reacts with PPi to regenerate GTP. In the reverse of Reaction ii, the enzyme catalyzes the transfer of the 5'-GMP from capped RNA to the Mr = 68,000 protein to form protein-guanylate complex. A divalent cation is required for both partial reactions. The Mr = 68,000 protein is presumed to be a subunit of the HeLa guanylyltransferase. This interpretation is consistent with the sedimentation coefficient of 4.2 S of the native enzyme. Preliminary studies of RNA guanylyltransferase from mouse myeloma tumors suggest a similar mechanism of transguanylylation involving a Mr = 68,000 protein-guanylate complex. These data, in conjunction with previous studies of vaccinia virus guanylyltransferase (Shuman, S., and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 187-191) suggests that covalent GMP-enzyme intermediates may be a general feature of the RNA capping reaction.     

The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity.

Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli. Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity. Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S. (1990) J. Biol. Chem. 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R. The individual subunits lacked methyltransferase activity when assayed alone. However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits. In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins. These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit. Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme.     

Yeast-based genetic system for functional analysis of poxvirus mRNA cap methyltransferase

Structural differences between poxvirus and human mRNA capping enzymes recommend cap formation as a target for antipoxviral drug discovery. Genetic and pharmacologic analysis of the poxvirus capping enzymes requires in vivo assays in which the readout depends on the capacity of the viral enzyme to catalyze cap synthesis. Here we have used the budding yeast Saccharomyces cerevisiae as a genetic model for the study of poxvirus cap guanine-N7 methyltransferase. The S. cerevisiae capping system consists of separate triphosphatase (Cet1), guanylyltransferase (Ceg1), and methyltransferase (Abd1) components. All three activities are essential for cell growth. We report that the methyltransferase domain of vaccinia virus capping enzyme (composed of catalytic vD1-C and stimulatory vD12 subunits) can function in lieu of yeast Abd1. Coexpression of both vaccinia virus subunits is required for complementation of the growth of abd1Delta cells. Previously described mutations of vD1-C and vD12 that eliminate or reduce methyltransferase activity in vitro either abolish abd1Delta complementation or elicit conditional growth defects. We have used the yeast complementation assay as the primary screen in a new round of alanine scanning of the catalytic subunit. We thereby identified several new amino acids that are critical for cap methylation activity in vivo. Studies of recombinant proteins show that the lethal vD1-C mutations do not preclude heterodimerization with vD12 but either eliminate or reduce cap methyltransferase activity in vitro.     

A Saccharomyces cerevisiae RNA 5'-triphosphatase related to mRNA capping enzyme

The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: the RNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Using computer homology searching, a S. cerevisiae gene was identified that encodes a protein resembling the C-terminal region of Cet1. Accordingly, we designated this gene CTL1 (capping enzyme RNAtriphosphatase-like 1). CTL1 is not essential for cell viability and no genetic or physical interactions with the capping enzyme genes were observed. The protein is found in both the nucleus and cytoplasm. Recombinant Ctl1 protein releases gamma-phosphate from the 5'-end of RNA to produce a diphosphate terminus. The enzyme is specific for polynucleotide RNA in the presence of magnesium, but becomes specific for nucleotide triphosphates in the presence of manganese. Ctl1 is the second member of the yeast RNA triphosphatase family, but is probably involved in an RNA processing event other than mRNA capping.     

Messenger RNA guanylyltransferase from Saccharomyces cerevisiae. Large scale purification, subunit functions, and subcellular localization

Messenger RNA capping enzyme (GTP:mRNA guanylyltransferase) purified from yeast Saccharomyces cerevisiae consisted of two polypeptides (45 and 39 kDa) and possessed two enzymatic activities, i.e. mRNA guanylyltransferase and RNA 5'-triphosphatase (Itoh, N., Mizumoto, K., and Kaziro, Y. (1984) J. Biol. Chem. 259, 13923-13929). In this paper, we describe an improved procedure suitable for the large scale purification of the enzyme. The steps include glass beads disruption of the cells and several ion-exchange and affinity column chromatographies. The enzyme was purified from kilogram quantities of yeast cells to apparent homogeneity. The purified enzyme had an approximate Mr of 180,000 and consisted of two heterosubunits of 80 and 52 kDa and had the same two enzymatic activities as above. We consider that this is the more intact form of the enzyme. Using the in situ assays on sodium dodecyl sulfate-polyacrylamide gels, RNA 5'-triphosphatase, and mRNA guanylyltransferase activities were located on the 80- and 52-kDa chains, respectively. In agreement with this, the 52-kDa enzyme-[32P]GMP complex was formed on incubation of the enzyme with [alpha-32P]GTP. Guinea pig antisera against purified yeast capping enzyme recognized both 80- and 52-kDa chains in Western blot analysis. The antibody did not cross-react with the enzymes from rat liver. Artemia salina, or vaccinia virus. Nuclear localization of the enzyme was demonstrated by immunofluorescence microscopy.     

RNA 5′-Triphosphatase, Nucleoside Triphosphatase, and Guanylyltransferase Activities of Baculovirus LEF-4 Protein

Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5′-triphosphatase that hydrolyzes the γ phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and P_i (K_m = 43 μM ATP; V_max = 30 s⁻¹) and GTP to GDP and P_i. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases.     

Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus

RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in cap formation. The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel. Interactions of a sulfate in the center of the tunnel with a divalent cation and basic amino acids projecting into the tunnel suggest a catalytic mechanism that is supported by mutational data. Discrete surface domains mediate Cet1p homodimerization and Cet1p binding to the guanylyltransferase component of the capping apparatus. The structure and mechanism of fungal RNA triphosphatases are completely different from those of mammalian mRNA capping enzymes. Hence, RNA triphosphatase presents an ideal target for structure-based antifungal drug discovery.     

Trypanosoma brucei RNA triphosphatase. Antiprotozoal drug target and guide to eukaryotic phylogeny.

The mRNA capping apparatus of the protozoan parasite Trypanosoma brucei consists of separately encoded RNA triphosphatase and RNA guanylyltransferase enzymes. The triphosphatase TbCet1 is a member of a new family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and the malaria parasite Plasmodium falciparum. The protozoal/fungal enzymes are structurally and mechanistically unrelated to the RNA triphosphatases of metazoans and plants. These results highlight the potential for discovery of broad spectrum antiprotozoal and antifungal drugs that selectively block the capping of pathogen-encoded mRNAs. We propose a scheme of eukaryotic phylogeny based on the structure of RNA triphosphatase and its physical linkage to the guanylyltransferase component of the capping apparatus.