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

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

The N-terminal 60 kDa (amino acids 1 to 545) of the D1 subunit of vaccinia virus mRNA capping enzyme is an autonomous bifunctional domain with triphosphatase and guanylyltransferase activities. We previously described two alanine cluster mutations, R77 to A (R77A)-K79A and E192A-E194A, which selectively inactivated the triphosphatase component. Here, we characterize the activities of 11 single alanine mutants-E37A, E39A, Q60A, E61A, T67A, T69A, K75A, R77A, K79A, E192A, and E194A-and a quadruple mutant in which four residues (R77, K79, E192, and E194) were replaced by alanine. We report that Glu-37, Glu-39, Arg-77, Glu-192, and Glu-194 are essential for gamma-phosphate cleavage. The five essential residues are conserved in the capping enzymes of Shope fibroma virus, molluscum contagiosum virus, and African swine fever virus. Probing the structure of D1(1-545) by limited V8 proteolysis suggested a bipartite subdomain structure. The essential residue Glu-192 is the principal site of V8 cleavage. Secondary cleavage by V8 occurs at the essential residue Glu-39. The triphosphatase-defective quadruple mutant transferred GMP to the triphosphate end of poly(A) to form a tetraphosphate cap structure, GppppA. We report that GppppA-capped RNA is a poor substrate for cap methylation by the vaccinia virus and Saccharomyces cerevisiae RNA (guanine-7) methyltransferases. The transcription termination factor activity of the D1-D12 capping enzyme heterodimer was not affected by mutations that abrogated ATPase activity. Thus, the capping enzyme is not responsible for the requirement for ATP hydrolysis during transcription termination.
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PMID:Structure-function analysis of the triphosphatase component of vaccinia virus mRNA capping enzyme. 937 57

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.
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PMID:Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II. 939 72

Previous studies demonstrated that the mammalian mRNA capping enzyme is a bifunctional enzyme containing RNA 5'-triphosphatase and mRNA guanylyl-transferase activities in a single polypeptide. In yeast, both the above activities are separated into two different subunits, alpha and beta, the genes for which we have cloned recently. It is thus interesting to compare the structural and functional relationships between the mammalian and yeast capping enzymes. Here we isolated two human cDNAs encoding mRNA capping enzymes termed hCAP1a and hCAP1b which encode 597 and 541 amino acids, respectively. They are different only at the region coding for the C-terminal portion of the enzyme. Comparison of the deduced amino acid sequences with other cellular and viral capping enzymes showed that all the regions conserved among mRNA guanylyltransferases are observed in our clones except one conserved C-terminal region which was absent in the hCAP1b protein. The purified recombinant hCAP1a gene product, hCAP1a, exhibited both RNA 5'-triphosphatase and mRNA guanylyltransferase activities. Deletion mutant analysis of hCAP1a showed that the N-terminal 213 amino acid fragment containing a tyrosine specific protein phosphatase motif catalyzed the RNA 5'-triphosphatase activity and the C-terminal 369 amino acid fragment exhibited the mRNA guanylyltransferase activity. On the other hand, hCAP1b showed RNA 5'-triphosphatase activity, but neither enzyme-GMP covalent complex formation nor cap structure formation was detected.
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PMID:Cloning and characterization of two human cDNAs encoding the mRNA capping enzyme. 947 87

We have conducted a biochemical and genetic analysis of mouse mRNA capping enzyme (Mce1), a bifunctional 597-amino acid protein with RNA triphosphatase and RNA guanylyltransferase activities. The principal conclusions are as follows: (i) the mammalian capping enzyme consists of autonomous and nonoverlapping functional domains; (ii) the guanylyltransferase domain Mce1(211-597) is catalytically active in vitro and functional in vivo in yeast in lieu of the endogenous guanylyltransferase Ceg1; (iii) the guanylyltransferase domain per se binds to the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD), whereas the triphosphatase domain, Mce1(1-210), does not bind to the CTD; and (iv) a mutation of the active site cysteine of the mouse triphosphatase elicits a strong growth-suppressive phenotype in yeast, conceivably by sequestering pre-mRNA ends in a nonproductive complex or by blocking access of the endogenous yeast triphosphatase to RNA polymerase II. These findings contribute to an emerging model of mRNA biogenesis wherein RNA processing enzymes are targeted to nascent polymerase II transcripts through contacts with the CTD. The phosphorylation-dependent interaction between guanylyltransferase and the CTD is conserved from yeast to mammals.
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PMID:The guanylyltransferase domain of mammalian mRNA capping enzyme binds to the phosphorylated carboxyl-terminal domain of RNA polymerase II. 954 88

Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5'-triphosphatase that hydrolyzes the gamma-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 microM gamma-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min-1 and converts ATP to ADP with Vmax of 8 min-1 and Km of 25 microM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5' ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase.
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PMID:Characterization of a baculovirus-encoded RNA 5'-triphosphatase. 969 98

The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5'-phosphatase. BVP sequentially removes gamma and beta phosphates from the 5' end of triphosphate-terminated RNA, leaving a 5'-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA.
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PMID:A protein tyrosine phosphatase-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activities. 970 57

We have characterized an essential Saccharomyces cerevisiae gene, CES5, that when present in high copy, suppresses the temperature-sensitive growth defect caused by the ceg1-25 mutation of the yeast mRNA guanylyltransferase (capping enzyme). CES5 is identical to CET1, which encodes the RNA triphosphatase component of the yeast capping apparatus. Purified recombinant Cet1 catalyzes hydrolysis of the gamma phosphate of triphosphate-terminated RNA at a rate of 1 s-1. Cet1 is a monomer in solution; it binds with recombinant Ceg1 in vitro to form a Cet1-Ceg1 heterodimer. The interaction of Cet1 with Ceg1 elicits >10-fold stimulation of the guanylyltransferase activity of Ceg1. This stimulation is the result of increased affinity for the GTP substrate. A truncated protein, Cet1(201-549), has RNA triphosphatase activity, heterodimerizes with and stimulates Ceg1 in vitro, and suffices when expressed in single copy for cell growth in vivo. The more extensively truncated derivative Cet1(246-549) also has RNA triphosphatase activity but fails to stimulate Ceg1 in vitro and is lethal when expressed in single copy in vivo. These data suggest that the Cet1-Ceg1 interaction is essential but do not resolve whether the triphosphatase activity is also necessary. The mammalian capping enzyme Mce1 (a bifunctional triphosphatase-guanylyltransferase) substitutes for Cet1 in vivo. A mutation of the triphosphatase active-site cysteine of Mce1 is lethal. Hence, an RNA triphosphatase activity is essential for eukaryotic cell growth. This work highlights the potential for regulating mRNA cap formation through protein-protein interactions.
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PMID:Genetic, physical, and functional interactions between the triphosphatase and guanylyltransferase components of the yeast mRNA capping apparatus. 971 Jun 3

mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present. Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD.
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PMID:Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain. 983 1

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.
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PMID:A Saccharomyces cerevisiae RNA 5'-triphosphatase related to mRNA capping enzyme. 1021 91

Saccharomyces cerevisiae RNA triphosphatase (Cet1p) and RNA guanylyltransferase (Ceg1p) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex. Cet1p binding to Ceg1p stimulates the guanylyltransferase activity of Ceg1p. Here we localize the guanylyltransferase-binding and guanylyltransferase-stimulation functions of Cet1p to a 21-amino acid segment from residues 239 to 259. The guanylyltransferase-binding domain is located on the protein surface, as gauged by protease sensitivity, and is conserved in the Candida albicans RNA triphosphatase CaCet1p. Alanine-cluster mutations of a WAQKW motif within this segment abolish guanylyltransferase-binding in vitro and Cet1p function in vivo, but do not affect the triphosphatase activity of Cet1p. Proteolytic footprinting experiments provide physical evidence that Cet1p interacts with the C-terminal domain of Ceg1p. Trypsin-sensitive sites of Ceg1p that are shielded from proteolysis when Ceg1p is bound to Cet1p are located between nucleotidyl transferase motifs V and VI.
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PMID:An essential surface motif (WAQKW) of yeast RNA triphosphatase mediates formation of the mRNA capping enzyme complex with RNA guanylyltransferase. 1057 65


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