Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
...
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.
...
PMID:The D1 and D12 subunits are both essential for the transcription termination factor activity of vaccinia virus capping enzyme. 774 34

The vaccinia virus D6R open reading frame encodes the small subunit of the heterodimeric vaccinia virus early transcription factor (VETF) that activates transcription of early genes in vitro. VETF binds early gene promoters and has a DNA-dependent ATPase activity that is essential for activation of transcription. To examine the relationship between the structure and function of VETF, we have localized the mutations in two temperature-sensitive viruses whose lesions previously were mapped to the D6R gene. For both mutants, a single G-to-A nucleotide change that would alter protein coding potential was identified. In mutant E93, the codon for alanine 25 was changed to that of threonine, and in mutant S4 the codon for valine 278 was replaced with that for methionine. The molecular phenotype of each mutant was assessed by expressing mutant transcription factors in HeLa cells by using a vaccinia virus-T7 system and characterizing the proteins' activities in vitro. The A25T mutant activated transcription to a lesser extent than wild-type VETF, and the V278M mutant had no demonstrable transcription factor activity. Both mutant proteins were shown to be defective for promoter binding, accounting for their impairment in transcription activation. The functional defects for both mutants were observed at permissive as well as nonpermissive temperatures. The mutant proteins retained ATPase activity but required higher DNA concentrations to activate the ATPase. These results indicate that the small subunit of VETF is essential for its promoter binding activity and likely contacts the promoter DNA. Immunoblotting experiments showed that the virion particles from the two mutant viruses contained about half the VETF of wild-type virus, suggesting that promoter binding may contribute to packaging of VETF into the virion particle. RNA polymerase, mRNA capping enzyme, and nucleoside triphosphate phosphohydrolase I were found at similarly reduced levels in the virion, indicating that packaging of some virion core enzymes may be interdependent.
...
PMID:Temperature-sensitive mutations in the gene encoding the small subunit of the vaccinia virus early transcription factor impair promoter binding, transcription activation, and packaging of multiple virion components. 813 39

The vaccinia virus mRNA capping enzyme is a heterodimeric protein containing subunits of 97 and 33 kDa, the products of genes D1R and D12L, respectively. The enzyme catalyzes the first three reactions in the mRNA cap formation pathway: mRNA triphosphatase, guanyltransferase and (guanine-7-)methyltransferase. The guanyltransferase reaction proceeds by way of a covalent enzyme GMP (E-GMP) intermediate (Shuman, S. and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 187-191) in which the GMP is linked to the large subunit through a lysine residue (Toyama, R., Mizumoto, K., Nakahara, Y., Tatsuno, T., and Kaziro, Y. (1983) Eur. J. Biochem. 2, 2195-2201; Roth, M. J., and Hurwitz, J. (1984) J. Biol Chem. 259, 13488-13494). In order to identify the map position of the guanyltransferase active site lysine residue, high specific activity [32P]E-GMP was prepared. Digestion of the E-GMP with hydroxylamine at pH 9.5 yielded a 31-kDa radioactive fragment derived from amino acids 1-273. Cleavage of E-GMP with cyanogen bromide produced a radioactive peptide of 14 kDa corresponding to amino acids 242-365. Lysine residues are found at positions 244 and 260. Staphylococcus aureus V8 protease digestion of cyanogen bromide-cleaved E-GMP yields a radioactive product of about 5 kDa in molecular mass corresponding to the peptide generated by cleavage at glutamic acid residues 253 and 297, demonstrating that lysine 260 is the site of linkage of GMP.
...
PMID:Identification of the vaccinia virus mRNA guanyltransferase active site lysine. 822 60

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.
...
PMID: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. 866 35

D1R1-545, an active subdomain of the large subunit of vaccinia virus mRNA capping enzyme possessing ATPase, RNA 5'-triphosphatase, and guanylyltransferase activities, was expressed in Escherichia coli and shown to be functionally equivalent to the heterodimeric enzyme (Myette, J. R., and Niles, E. G. (1996) J. Biol. Chem. 271, 11936-11944). A detailed characterization of the phosphohydrolytic activities of D1R1-545 demonstrates that, in addition to ATPase and RNA 5'-triphosphatase activities, the capping enzyme also possesses a general nucleoside triphosphate phosphohydrolase activity that lacks a preference for the nucleoside base or sugar. Nucleoside triphosphate and mRNA saturation kinetics are markedly different, with RNA exhibiting a Km and turnover number 100- and 10-fold less, respectively, than those values measured for any NTP. The linear competitive inhibition of RNA 5'-triphosphatase activity by ATP, and the relative manner by which both ATPase and RNA 5'-triphosphatase activities are inhibited by specific oligonucleotides, kinetically demonstrate that each activity is carried out at a common active site. Direct UV photo-cross-linking of either 32P-radiolabeled ATP or 23-mer triphosphorylated RNA, followed by cyanogen bromide cleavage of the photo-linked enzyme, localizes the major binding site for both ATP and RNA to a region between amino acids 1 and 221. The inability of ATP to competitively inhibit either E approximately GMP formation or the transfer of GMP to RNA kinetically differentiates the phosphohydrolase active site from the guanylyltransferase active site.
...
PMID:Characterization of the vaccinia virus RNA 5'-triphosphatase and nucleotide triphosphate phosphohydrolase activities. Demonstrate that both activities are carried out at the same active site. 866 36

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.
...
PMID:Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA capping enzyme. 870 42

The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.
...
PMID:Localization and in vitro mutagenesis of the active site in the Saccharomyces cerevisiae mRNA capping enzyme. 872 Jan 51

Temperature-sensitive mutations (ts10, ts18, and ts39) of the vaccinia virus RNA helicase nucleoside triphosphate phosphohydrolase II (NPH-II) result in the production of noninfectious progeny virions at the restrictive temperature. The noninfectious mutant particles contain the wild-type complement of virion core and envelope polypeptides, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of Western blot (immunoblot) analysis indicate that these particles lack NPH-II, whereas other enzymatic components of the virus core are present. These components include the following: DNA-dependent RNA polymerase subunits rpo147, rpo132, rpo94, rpo35, rpo30, rpo22, and rpo18; early transcription initiation factor subunits A8 and D6; mRNA capping enzyme subunits D1 and D12; RNA cap 2'-O-methyltransferase; A18 DNA helicase; DNA-dependent ATPase NPH-I; and DNA topoisomerase. Although RNA polymerase is encapsidated by the mutant viruses, mRNA synthesis in vitro by permeabilized mutant virions is only 5 to 20% that of the wild-type virus, as judged by nucleoside monophosphate incorporation into acid-insoluble material. Moreover, the transcripts synthesized by the mutant particles are longer than normal and remain virion associated. Transcription initiation by mutant virions occurs accurately at an endogenous genomic promoter, albeit at reduced levels (1 to 7%) compared with that of wild-type virions. In contrast, extracts of the mutant virions catalyze the wild-type level of transcription from an exogenous template containing an early promoter. We conclude that NPH-II is required for early mRNA synthesis uniquely in the context of the virus particle. Possible roles in transcription termination and RNA transport are discussed.
...
PMID:Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II are defective in early transcription. 897 Sep 79

The yeast Saccharomyces cerevisiae mRNA capping enzyme is composed of two subunits of alpha (52 kDa, mRNA guanylyltransferase) and beta (80 kDa, RNA 5'-triphosphatase). We have isolated the alpha subunit gene (CEG1) by immunological screening. In this report, with the aid of partial amino acid sequences of purified yeast capping enzyme, we isolated the gene, designated CET1, encoding the S. cerevisiae capping enzyme beta subunit. Amino acid sequence analysis revealed that the gene encodes for 549 amino acids with a calculated M(r) of 61,800 which is unexpectedly smaller than the size estimated by SDS-PAGE. Gene disruption experiment showed that CET1 is essential for yeast cell growth. The purified recombinant CET1 gene product, Cet1, exhibited an RNA 5'-triphosphatase activity which specifically removed the gamma-phosphate from the triphosphate-terminated RNA substrate, but not from nucleoside triphosphates, confirming the identity of the gene. Interaction between the Cet1 and the Ceg1 was also studied by the West-Western procedure using recombinant Ceg1-[32P]GMP as probe.
...
PMID:Isolation and characterization of the yeast mRNA capping enzyme beta subunit gene encoding RNA 5'-triphosphatase, which is essential for cell viability. 934 80


<< Previous 1 2 3 4 5 Next >>

---