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At least 19 recordsLinked to original sources
PubMed1976

mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites.

Characterization of the donor and acceptor specificities of mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase isolated from vaccinia virus cores has enabled us to discriminate between alternative reaction sequences leading to the formation of the 5'-terminal m7G(5')pppN-structure. The mRNA guanylyltransferase catalyzes the transfer of a residue of GMP from GTP to acceptors which possess a 5'-terminal diphosphate. A diphosphate-terminated polyribonucleotide is preferred to a mononucleoside diphosphate as an acceptor suggesting that the guanylyltransferase reaction occurs after initiation of RNA synthesis. Although all of the homopolyribonucleotides tested (pp(A)n, pp(G)n, pp(I)n, pp(U)n, and pp(C)n) are acceptors for the mRNA guanylyltransferase indicating lack of strict sequence specificity, those containing purines are preferred. Only GTP and dGTP are donors in the reaction; 7-methylguanosine (m7G) triphosphate specifically is not a donor indicating that guanylylation must precede guanine-7-methylation. The preferred acceptor of the mRNA (guanine-7-)-methyltransferase is the product of the guanylyltransferase reaction, a polyribonucleotide with the 5'-terminal sequence G(5')pppN-. The enzyme can also catalyze, but less efficiently methylation of the following: dinucleoside triphosphates with the structure G(5')pppN, GTP, dGTP, ITP, GDP, GMP, and guanosine. The enzyme will not catalyze the transfer of methyl groups to ATP, XTP, CTP, UTP, or to guanosine-containing compounds with phosphate groups in either positions 2' or 3' or in 3'-5' phosphodiester linkages. The latter specificity provides an explanation for the absence of internal 7-methylguanosine in mRNA. In the presence of PPi, the mRNA guanylyltransferase catalyzes the pyrophosphorolysis of the dinucleoside triphosphate G(5')pppA, but not of m7G(5')pppA. Since PPi is generated in the process of RNA chain elongation, stabilization of the 5'-terminal sequences of mRNA is afforded by guanine-7-methylation.

Guanosine Triphosphate↗
PubMed1975

Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions.

The sequences m7G(5')pppGm-and m7G(5')pppAm-are located at the 5' termini of vaccinia mRNAs. Two novel enzymatic activities have been purified from vaccinia virus cores which modify the 5' terminus of unmethylated mRNA. One activity transfers GMP from GTP to mRNA and is designated a GTP: mRNA guanylyltransferase. The second activity transfers a methyl group from S-adenosylmethionine to position 7 of the added guanosine and is designated a S-adenosylmethionine: mRNA (guanine-7-)methyltransferase. Advantage was taken of the selective binding of these activities to homopolyribonucleotides relative to DNA to achieve a 200-fold increase in specific activity. The guanylyl- and methyltransferase remained inseparable during chromatography on DNA-agarose, poly(U)-Sepharose, poly(A)-Sepharose, and Sephadex G-200 and during sedimentation through sucrose density gradients suggesting they were associated. A Stokes radius of 5.0 nm, an S20,w of 6.0 and a molecular weight of 127,000 were obtained by gel filtration on Sephadex G-200 and sedimentation in sucrose density gradients. Under denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis two major polypeptides were detected in purified enzyme preparations. Their molecular weights of 95,000 and 31,400 suggested they were polypeptide components of the 127,000 molecular weight enzyme system.

Guanine Nucleotides↗
PubMed1975

Modification of RNA by mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions.

A purified enzyme system isolated from vaccinia virus cores has been shown to modify the 5' termini of viral mRNA and synthetic poly(A) and poly(G) to form the structures m7G(5')pppA- and m7G(5')pppG-. The enzyme system has both guanylyltransferase and methyltransferase activities. The GTP:mRNA guanylyltransferase activity incorporates GMP into the 5' terminus via a 5'-5' triphosphate bond. The properties of this reaction are: (a) of the four nucleoside triphosphates only GTP is a donor, (b) mRNA with two phosphates at the 5' terminus is an acceptor while RNA with a single 5'-terminal phosphate is not, (c) Mg2+ is required, (d) the pH optimum is 7.8, (e) PP1 is a strong inhibitor, and (f) the reverse reaction, namely the formation of GTP from PP1 and RNA containing the 5'-terminal structure G(5')pppN-, readily occurs. The S-adenosylmethionine:mRNA(guanine-7-)methyltransferase activity catalyzes the methylation of the 5'-terminal guanosine. This reaction exhibits the following characteristics: (a) mRNA with the 5'-terminal sequences G(5')pppA- and G(5')pppG- are acceptors, (b) only position 7 of the terminal guanosine is methylated; internal or conventional 5'-terminal guanosine residues are not methylated, (c) the reaction is not dependent upon GTP or divalent cations, (d) optimal activity is observed in a broad pH range around neutrality, (e) the reaction is inhibited by S-adenosylhomocysteine. Both the guanylyltransferase and methyltransferase reactions exhibit bisubstrate kinetics and proceed via a sequential mechanism. The reactions may be summarized: (see article).

Cations, Divalent↗
PubMed1977

Synthesis of mRNA guanylyltransferase and mRNA methyltransferases in cells infected with vaccinia virus.

Guanylyltransferase and methyltransferases that modify the 5'-terminals of viral mRNA's to form the structures m7G(5')pppAm- and m7G(5')pppGm- appear to be synthesized afte- vaccinia virus infection of HeLa cells. Elevations in these enzyme activities were detected within 1 h after virus inoculation and increased 15- to 30-fold by 4 to 10 h. Increases in the guanylyl- and methyltransferase activities were prevented by cycloheximide, an inhibitor of protein synthesis, but not by cytosine arabinoside, an inhibitor of DNA synthesis. The latter results suggest that the mRNA guanylyl- and methyltransferases are "early" or prereplicative viral gene products. The guanylyltransferase and two methyltransferases, a guanine-7-methyltransferase and nucleoside-2'-methyltransferase, were isolated by column chromatography from infected cell extracts and found to have properties similar or identical to those of the corresponding enzyme previously isolated from vaccinia virus cores. In contrast, enzymes with these properties could not be isolated from uninfected cells.

Cycloheximide↗
PubMed1982

Purification of mRNA guanylyltransferase from calf thymus.

mRNA guanylyltransferase has been extensively purified from calf thymus. A GTP-binding assay was used based on the observations by Shuman and Hurwitz (1981) and Venkatesan and Moss (1982) that vaccinia virus and HeLa cell mRNA guanylyltransferases bind the GMP moiety from GTP in the absence of an acceptor RNA. The mol. wt. of the purified enzyme from calf thymus, estimated by polyacrylamide gel electrophoresis in the presence of SDS, is 65 000. The major protein in the purified enzyme fraction comigrates with the peptide labelled with GMP. Based on scans of silver-stained polyacrylamide gels, mRNA guanylyltransferase constitutes greater than 50% of the protein in these fractions. The enzyme catalyzed the guanylylation at the 5' end of poly(A) with a mixture of diphosphate and triphosphate ends. No evidence was obtained for a direct interaction between mRNA guanylyltransferase and RNA polymerase B (II).

Animals↗
PubMed1980

Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei.

GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of a GMP residue from GTP to the 5' end of RNA to form a cap structure identified as G(5')pppN-, has been isolated from HeLa cell nuclei. The enzyme has been purified approximately 1000-fold and separated by column chromatography (using DEAE-cellulose, phosphocellulose, Cibacron blue-agarose, and GTP-agarose) from a variety of other activities, including RNA triphosphatase and mRNA (guanine-7)methyltransferase. The reaction product was identified by its resistance to Penicillium nuclease and alkaline phosphatase, sensitivity to venom phosphodiesterase, and electrophoretic and chromatographic mobilities relative to authentic standards. Optimal enzyme activity was obtained at pH 7.5 in the presence of Mn2+ or Mg2+, GTP, and an appropriate acceptor polyribonucleotide. The enzyme was inhibited by elevated concentrations of salt and by sulfhydryl-binding reagents but was unaffected by S-adenosylmethionine or S-adenosylhomocysteine. A molecular weight of 48,500 was estimated by sucrose gradient centrifugation of purified enzyme.

Cell Nucleus↗
PubMed1997

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II.

5'-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5'-triphosphatase activity and catalyzed formation of RNA 5'-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5'-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.

Amino Acid Sequence↗
PubMed1983

Mechanism of the mRNA guanylyltransferase reaction: isolation of N epsilon-phospholysine and GMP (5' leads to N epsilon) lysine from the guanylyl-enzyme intermediate.

The mRNA capping reaction catalyzed by rat liver mRNA guanylyltransferase proceeds through an enzyme-GMP intermediate in which GMP is linked to the enzyme by a phosphoamide linkage. The studies described here show that GMP is bound to the epsilon-amino group of lysine of rat liver guanylyltransferase. The enzyme-[32P]GMP intermediate was digested with pronase to a [32P]GMP-peptide which was then converted to [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. After alkaline hydrolysis of the [32P]phosphoryl-peptide, the major radioactive product co-electrophoresed with the authentic N epsilon-phospholysine on DEAE-cellulose paper. Neither [32P]Nimid-phosphohistidine nor Nguanido-phosphoarginine was detected in the hydrolysates. Furthermore, formation of N epsilon-guanylyl-lysine linkage on the enzyme was more directly shown by isolation of [32P]GMP(5' leads to N epsilon)lysine when the steps of periodate oxidation and beta-elimination were omitted. The results indicate that the nucleophile in the guanylyltransferase to which the guanylyl residue is linked is the epsilon-amino group of a lysine residue. [32P]Phosphoryl-lysine was also isolated from the vaccinia virus capping enzyme-[32P]GMP intermediate. Guanylyltransferase from HeLa cells, wheat germ, Artemia salina and yeast also formed the enzyme-GMP complex and, from the stability of the complex, the linkage between the enzyme and GMP was suggested to be a phosphoamide.

Animals↗
PubMed2003

Identification of two histidines necessary for reovirus mRNA guanylyltransferase activity.

Grass carp reovirus, a segmented double-stranded RNA virus, is a member of the genus aquareovirus in the Reoviridae family. Grass carp reovirus VP1 was shown to be an mRNA guanylyltransferase. The enzyme demonstrated maximum activity <or= pH 6.0. This low pH maximum is conserved among the known guanylyltransferases of the Reoviridae family, but is not a property of the KxDG guanylyltransferases. The positive effect of low pH was detected for both autoguanylylation and GMP transfer, the two steps in the guanylyltransferase reaction. The effect of pH on enzymatic activity suggested that histidine protonation is responsible for the observed increase in guanylyltransferase activity. Mutagenesis of the two histidines conserved among the orthoreovirus and aquareovirus guanylyltransferases demonstrated that they are necessary for activity.

Amino Acid Sequence↗
PubMed1987

Complete nucleotide sequence of reovirus L2 gene and deduced amino acid sequence of viral mRNA guanylyltransferase.

Reovirus mRNAs synthesized by the virion-associated RNA polymerase contain a 5'-terminal cap that is added to nascent transcripts by polypeptide lambda 2, a structural component of virions encoded by double-stranded RNA genome segment L2. The complete, 3916-nucleotide sequence of a full-length reovirus type 3 L2 DNA clone was determined by the dideoxy chain terminator method. The sequence has a single long open reading frame extending from the second A-T-G at nucleotide 14 to a termination codon at position 3881. On this basis, the 1289-amino acid sequence of polypeptide lambda 2, the reovirus mRNA guanylyltransferase, was deduced and compared to other GTP-binding proteins. Two different, lysine-containing lambda 2 peptide sequences closely resemble predicted amino acid stretches in vaccinia virus guanylyltransferase and potentially form part of active sites in the viral mRNA capping enzymes.

Amino Acid Sequence↗
PubMed1982

Reaction mechanism of mRNA guanylyltransferase from rat liver: isolation and characterization of a guanylyl-enzyme intermediate.

Rat liver RNA guanylyltransferase catalyzes a GTP-PPi exchange reaction in the absence of acceptor RNA [Mizumoto, K. & Lipmann, F. (1979) Proc. Natl. Acad. Sci. USA 76, 4961-4965] suggesting that the reaction proceeds through the formation of a covalent guanylylated intermediate. We now present more direct evidence for the existence of the enzyme-GMP intermediate: (i) the enzyme-[32P]GMP intermediate was formed on incubation of rat liver guanylyltransferase with [alpha-32P]GTP and migrated as a single radioactive band with Mr 69,000 on NaDodSO4/polyacrylamide gel electrophoresis, and (ii) the intermediate isolated on gel filtration can transfer its GMP moiety to ppGpCpC-poly(A2,U2,G) to form the capped RNA molecule or it can react with PPi to regenerate GTP. The formation of the intermediate was dependent on Mg2+ and was strongly inhibited by PPi. The addition of pyrophosphatase markedly increased the amount of the intermediate complex. On blue dextran-Sepharose affinity column chromatography, the activity of guanylyltransferase to form an enzyme-[32P]GMP intermediate comigrated with activities of cap formation and GTP-PPi exchange. A phosphoamide type linkage between GMP and enzyme is suggested by its acidlabile and alkali-stable nature and also by the susceptibility to acidic hydroxylamine. These results indicate that the reaction catalyzed by rat liver guanylyltransferase occurs through the following two partial steps: (i) E + GTP in equilibrium E-pG + PPi; and (ii) E-pG + ppN .....leads to GpppN .....+ E.

Animals↗
PubMed1978

Purification of mRNA guanylyltransferase from vaccinia virions.

GTP:RNA guanylyltransferase, the enzyme which catalyzes the guanylylation of the 5' termini of viral mRNAs, has been isolated and purified approximately 10,000-fold from cores of vaccinia virus. S-adenosyl-methionine:mRNA (guanine-7)-methyltransferase copurified with guanylyltransferase activity through chromatography on DNA agarose, phosphocellulose, and centrifugation in glycerol gradients, suggesting that the two activities are closely associated. The molecular weight of native guanylyltransferase- and 7-methyltransferase-associated activities was approximately 120,000 as determined by glycerol gradient centrifugation. Guanylytransferase purified by electrophoresis on polyacrylamide gels at pH 4.5 lacked 7-methyltransferase activity. Analysis by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels of electrophoretically purified native guanylyltransferase showed the presence of one major band of polypeptide which had a molecular weight of approximately 59,000.

Guanine Nucleotides↗
PubMed1980

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.

Cations, Divalent↗
PubMed1999

The Candida albicans gene for mRNA 5-cap methyltransferase: identification of additional residues essential for catalysis.

The 5'-cap structure of eukaryotic mRNA is methylated at the terminal guanosine by RNA (guanine-N7-)-methyltransferase (cap MTase). Saccharomyces cerevisiae ABD1 (ScABD1) and human hMet (also called CMT1) genes are responsible for this enzyme. The ABD1 homologue was cloned from the pathogenic fungus Candida albicans and named C. albicans ABD1 (CaABD1). When expressed as a fusion with glutathione S-transferase (GST), CaAbd1p displayed cap MTase activity in vitro and rescued S. cerevisiae abd1delta null mutants, indicating that CaABD1 specifies an active cap MTase. Although the human cap MTase binds to the human capping enzyme (Hce1p), which possesses both mRNA guanylyltransferase (mRNA GTase) and mRNA 5'-triphosphatase (mRNA TPase) activities, yeast two-hybrid analysis demonstrated that in yeast neither mRNA GTase nor mRNA TPase physically interacted with the Abd1 protein. Comparison of the amino acid sequences of known and putative cap MTases revealed a highly conserved amino acid sequence motif, Phe/Val-Leu-Asp/Glu-Leu/Met-Xaa-Cys-Gly-Lys-Gly-Gly-Asp-Leu-Xaa-Lys, which encompasses the sequence motif characteristic of divergent methyltransferases. Mutations in CaAbd1p of leucine at the second and the twelfth positions (so far uncharacterized) to alanine severely diminished the enzyme activity and the functionality in vivo, whereas those of leucine at the fourth, cysteine at the sixth, lysine at the eighth, and glycine at the tenth positions did not. Furthermore, valine substitution for the twelfth, but not for the second, leucine in that motif abolished the activity and functionality of CaAbd1p. Thus, it appears that leucine at the second and the twelfth positions in the motif, together with a previously identified acidic residue in the third, glycine at the sixth and glutamic acid at the eleventh positions, play important roles in the catalysis, and that side chain length is crucial for the activity at the twelfth position in the motif.

Amino Acid Motifs↗
PubMed1982

Eukaryotic mRNA capping enzyme-guanylate covalent intermediate.

Incubation of HeLa cell mRNA guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) with [alpha-32P]GTP and a divalent cation in the absence of an RNA acceptor results in the formation of a covalent enzyme-guanylate complex. The complex, after purification by phosphocellulose chromatography, can transfer its bound GMP moiety to pyrophosphate, regenerating GTP, or to the 5'-diphosphate end of poly(A), forming a cap structure G(5')pppA(pA)n. The GMP-polypeptide has a molecular weight of 65,000 and is stable to heating in the presence of sodium dodecyl sulfate. On the basis of the alkali-stable and acid-labile nature of the bond and its susceptibility to nucleophilic attack by hydroxylamine at low pH, the GMP-polypeptide linkage appears to be a phosphoamine bond. After digestion with trypsin, a single GMP-peptide was resolved by two dimensional electrophoresis and chromatography.

Guanine Nucleotides↗
PubMed1998

Isolation and characterization of the Candida albicans gene for mRNA 5'-triphosphatase: association of mRNA 5'-triphosphatase and mRNA 5'-guanylyltransferase activities is essential for the function of mRNA 5'-capping enzyme in vivo.

The amino acid sequence of the Saccharomyces cerevisiae mRNA 5'-triphosphatase (TPase) diverges from those of higher eukaryotes. In order to confirm the sequence divergence of TPases in lower and higher eukaryotes, the Candida albicans gene for TPase was identified and characterized. This gene designated CaCET1 (C. albicans mRNA 5'-capping enzyme triphosphatase 1) has an open reading frame of 1.5 kb, which can encode a 59-kDa protein. Although the N-terminal one-fifth of S. cerevisiae TPase (ScCet1p) is missing in CaCet1p, CaCet1p shares significant sequence similarity with ScCet1p over the entire region of the protein; the recombinant CaCet1p, which was expressed as a fusion protein with glutathione S-transferase (GST), displayed TPase activity in vitro. CaCET1 rescued CET1-deficient S. cerevisiae cells when expressed under the control of the ADH1 promoter, whereas the human capping enzyme derivatives that are active for TPase activity but defective in mRNA 5'-guanylyltransferase (GTase) activity did not. Yeast two-hybrid analysis revealed that C. albicans Cet1p can bind to the S. cerevisiae GTase in addition to its own partner, the C. albicans GTase. In contrast, neither the full-length human capping enzyme nor its TPase domain interacted with the yeast GTase. These results indicate that the failure of the human TPase activity to complement an S. cerevisiae cet1delta null mutation is attributable, at least in part, to the inability of the human capping enzyme to associate with the yeast GTase, and that the physical association of GTase and TPase is essential for the function of the capping enzyme in vivo.

Acid Anhydride Hydrolases↗