Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.25 (triphosphatase)
 1,529 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The highly purified yeast mRNA capping enzyme is composed of two separate chains of 52 (alpha) and 80 kDa (beta), responsible for the activities of mRNA guanylyltransferase and RNA 5'-triphosphatase, respectively (Itoh, N., Yamada, H., Kaziro, Y., and Mizumoto, K. (1987) J. Biol. Chem. 262, 1989-1995). The gene encoding the mRNA guanylyltransferase subunit (alpha subunit), CEG1, has been isolated by immunological screening of a yeast genomic expression library in lambda gt11 with polyclonal antibodies directed against purified yeast capping enzyme. The identity of CEG1 was confirmed by epitope selection and by expressing the gene in Escherichia coli to give a catalytically active mRNA guanylyltransferase. The gene is present in one copy per haploid genome, and encodes a polypeptide of 459 amino acid residues. From its primary structure as well as its mRNA size, it was concluded that the alpha and the beta subunits of yeast mRNA capping enzyme are encoded by two separate genes, not as a fused protein. CEG1 is located on the chromosome VII by a pulse-field gel electrophoresis. Gene disruption experiment indicated that CEG1 is essential for the growth of yeast. We have also found another open reading frame (ORF2) which lies in close proximity to CEG1 in our clones and encodes a 450 amino acid-polypeptide of yet unknown function.
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PMID:mRNA capping enzyme. Isolation and characterization of the gene encoding mRNA guanylytransferase subunit from Saccharomyces cerevisiae. 131 57

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.
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PMID:Methyltransferase and subunit association domains of vaccinia virus mRNA capping enzyme. 132 1

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.
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PMID:The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity. 132 2

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.
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PMID:Catalytic activity of vaccinia mRNA capping enzyme subunits coexpressed in Escherichia coli. 216 22

RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7)-methyltransferase, and transcription termination factor activities are associated with the mRNA capping enzyme from vaccinia virus. Purified vaccinia capping enzyme is a 6.5 S protein containing two subunits of Mr = 95,000 and Mr = 31,000. Although the RNA guanylyltransferase domain has been localized to the large subunit by virtue of the formation of a Mr = 95,000 covalent protein-GMP intermediate, the location of other functional domains within the protein and the catalytic role of individual subunits remain unclear. In the present study, limited proteolysis with trypsin was shown to convert the vaccinia capping enzyme into a form capable of generating a Mr = 59,000 enzyme-GMP complex. Purification of the trypsinized enzyme by glycerol gradient sedimentation resulted in the isolation of a 4.2 S fragment of the large subunit that retains RNA triphosphatase and RNA guanylyltransferase activities. This derivative, containing little or no small subunit (or fragments thereof), has lost the ability to catalyze methyl group transfer and to mediate transcription termination in vitro. Residual methyltransferase activity was found associated with a minor 5.2 S tryptic product that cosediments with a Mr = 21,000 fragment of the small enzyme subunit. A model for the organization of functional domains within the capping enzyme is suggested.
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PMID:Functional domains of vaccinia virus mRNA capping enzyme. Analysis by limited tryptic digestion. 254 18

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.
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PMID:Messenger RNA guanylyltransferase from Saccharomyces cerevisiae. Large scale purification, subunit functions, and subcellular localization. 302 58

Highly purified mRNA-capping enzyme from Saccharomyces cerevisiae catalyzes (a) removal of the gamma-phosphoryl group from the 5'-end of the newly formed mRNA and (b) guanylylation of the resulting diphosphoryl end. Characteristics of the two reactions catalyzed by this enzyme are studied. Guanylyltransferase is most active at pH 7.0 in the presence of 3 mM Mg2+, and utilizes GTP as a guanylyl donor with an apparent Km of 5 microM, and ppGCC (A2, U2, G)n as a guanylyl acceptor with two Km values of 0.5 and 4 microM. It catalyzes GTP-PPi exchange in the absence of the acceptor RNA, and forms a covalent enzyme-GMP intermediate having Mr = 45,000 in sodium dodecyl sulfate gel electrophoresis. RNAs with 5'-diphosphoryl as well as 5'-triphosphoryl ends are capped, while mononucleotides such as GDP and ppGp are inert. Since guanylyltransferase can utilize ppGpC and ppGpCpC as acceptors, the presence of at least one phosphodiester bond seems to be sufficient for the acceptor activity. However, oligonucleotides of longer chain length are preferred. RNA 5'-triphosphatase associated with the purified enzyme requires Mg2+ and exhibits a broad pH optimum from 6.5 to 8.5, and an apparent Km value for pppA-terminated poly(A) is 1.4 microM. The enzyme is specific for the gamma-phosphoryl group at the 5'-terminus of RNA and does not hydrolyze ATP. It can hydrolyze the gamma-phosphoryl group of pppGp, but the RNA substrates with longer chain length are preferred.
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PMID:Messenger RNA guanlyltransferase from Saccharomyces cerevisiae. II. Catalytic properties. 609 33

An RNA 5'-triphosphatase activity hydrolyzing gamma-phosphate from pppN-RNA was found to be associated with mRNA guanylyltransferase partially purified from rat liver nuclei. The activity specifically removed 32P as inorganic phosphate from [gamma-32P]pppA(pA)n, but not from [beta-32P]pppA(pA)n or from [gamma-32P]ATP. Free SH group(s) were required for its activity, and the reaction was inhibited by N-ethylmaleimide. Divalent cations were not required, but were rather inhibitory for the reaction. The RNA 5'-triphosphatase activity could not be separated from the guanylyltransferase activity through successive chromatographies on Sephadex G-150, CM-Sephadex and blue dextran-Sepharose columns. Both activities remained physically associated during sedimentation in glycerol density gradients after high salt treatment. The heat stability of the RNA 5'-triphosphatase activity was almost identical with that of the guanylyltransferase activity. These results indicate that the 69000 mol. wt. protein purified from rat liver nuclei as guanylyltransferase possesses both mRNA capping and RNA 5'-triphosphatase activities.
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PMID:Association of an RNA 5'-triphosphatase activity with RNA guanylyltransferase partially purified from rat liver nuclei. 613 53

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7) methyltransferase, and transcription termination factor activities. The protein is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The capping reaction entails transfer of GMP from GTP to the 5'-diphosphate end of mRNA via a covalent enzyme-(lysyl-GMP) intermediate. The active site is situated at Lys-260 of the D1 subunit within a sequence element, KxDG (motif I), that is conserved in the capping enzymes from yeasts and other DNA viruses and at the active sites of covalent adenylylation of RNA and DNA ligases. Four additional sequence motifs (II to V) are conserved in the same order and with similar spacing among the capping enzymes and several ATP-dependent ligases. The relevance of these common sequence elements to the RNA capping reaction was addressed by mutational analysis of the vaccinia virus D1 protein. Nine alanine substitution mutations were targeted to motifs II to V. Histidine-tagged versions of the mutated D1 polypeptide were coexpressed in bacteria with the D12 subunit, and the His-tagged heterodimers were purified by Ni affinity and phosphocellulose chromatography steps. Whereas each of the mutated enzymes retained triphosphatase, methyltransferase, and termination factor activities, six of nine mutant enzymes were defective in some aspect of transguanylylation. Individual mutations in motifs III, IV, and V had distinctive effects on the affinity of enzyme for GTP, the rate of covalent catalysis (EpG formation), or the transfer of GMP from enzyme to RNA. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a conserved structural basis for covalent nucleotidyl transfer.
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PMID:Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA. 756 75

Transcription termination by vaccinia virus RNA polymerase during synthesis of early mRNAs requires a virus-encoded termination factor (VTF). VTF is but one of many activities associated with the vaccinia virus mRNA capping enzyme, a heterodimer of 95- and 33-kDa subunits encoded by the D1 and D12 genes, respectively. Although the three catalytic domains involved in cap formation have been assigned to individual subunits or portions thereof, the structural requirements for VTF activity are unknown. We now report that both full-length subunits are required for transcription termination. The 844-amino acid D1 subunit by itself, which is fully active in triphosphatase and guanylyltransferase functions, has no demonstrable VTF activity in vitro. Neither does the D12 subunit by itself. The heterodimeric methyltransferase domain of D1 (residues 498 to 844) and D12 subunits also has no VTF activity. VTF is not affected by a K-to-M mutation of the guanylyltransferase active site at position 260 (K260M) that abolishes enzyme-GMP complex formation or by a H682A/Y683A double mutation of the D1 subunit, which abrogates methyltransferase activity. Thus, the structural requirements for termination are distinct from those for nucleotidyl transfer and methyl transfer.
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PMID:The D1 and D12 subunits are both essential for the transcription termination factor activity of vaccinia virus capping enzyme. 774 34


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