Página 1 dos resultados de 422 itens digitais encontrados em 0.006 segundos

Helicase and Capping Enzyme Active Site Mutations in Brome Mosaic Virus Protein 1a Cause Defects in Template Recruitment, Negative-Strand RNA Synthesis, and Viral RNA Capping

Ahola, Tero; den Boon, Johan A.; Ahlquist, Paul
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2000 EN
Relevância na Pesquisa
46.73%
Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1, encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants...

RNA-Binding and Capping Activities of Proteins in Rotavirus Open Cores

Patton, John T.; Chen, Dayue
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/1999 EN
Relevância na Pesquisa
36.65%
Guanylyltransferases are members of the nucleotidyltransferase family and function in mRNA capping by transferring GMP to the phosphate end of nascent RNAs. Although numerous guanylyltransferases have been identified, studies which define the nature of the interaction between the capping enzymes of any origin and their RNA substrates have been limited. Here, we have characterized the RNA-binding activity of VP3, a minor protein component of the core of rotavirions that has been proposed to function as the viral guanylyltransferase and to direct the capping of the 11 transcripts synthesized from the segmented double-stranded RNA (dsRNA) genome of these viruses. Gel shift analysis performed with disrupted (open) virion-derived cores and virus-specific RNA probes showed that VP3 has affinity for single-stranded RNA (ssRNA) but not for dsRNA. While the ssRNA-binding activity of VP3 was found to be sequence independent, the protein does exhibit preferential affinity for uncapped over capped RNA. Like the RNA-binding activity, RNA capping assays performed with open cores indicates that the guanylyltransferase activity of VP3 is nonspecific and is able to cap RNAs initiating with a G or an A residue. These data establish that all three rotavirus core proteins...

The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA

Zhou, Meisheng; Deng, Longwen; Kashanchi, Fatah; Brady, John N.; Shatkin, Aaron J.; Kumar, Ajit
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
36.6%
The HIV type 1 (HIV-1) Tat protein stimulates transcription elongation by recruiting P-TEFb (CDK9/cyclin T1) to the transactivation response (TAR) RNA structure. Tat-induced CDK9 kinase has been shown to phosphorylate Ser-5 of RNA polymerase II (RNAP II) C-terminal domain (CTD). Results presented here demonstrate that Tat-induced Ser-5 phosphorylation of CTD by P-TEFb stimulates the guanylyltransferase activity of human capping enzyme and RNA cap formation. Sequential phosphorylation of CTD by Tat-induced P-TEFb enhances the stimulation of human capping enzyme guanylyltransferase activity and RNA cap formation by transcription factor IIH-mediated CTD phosphorylation. Using an immobilized template assay that permits isolation of transcription complexes, we show that Tat/TAR-dependent phosphorylation of RNAP II CTD stimulates cotranscriptional capping of HIV-1 mRNA. Upon transcriptional induction of latently infected cells, accumulation of capped transcripts occurs along with Ser-5-phosphorylated RNAP II in the promoter proximal region of the HIV-1 genome. Therefore, these observations suggest that Tat/TAR-dependent phosphorylation of RNAP II CTD is crucial not only in promoting transcription elongation but also in stimulating nascent viral RNA capping.

Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II

Mandal, Subhrangsu S.; Chu, Chun; Wada, Tadashi; Handa, Hiroshi; Shatkin, Aaron J.; Reinberg, Danny
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
36.5%
Capping of the 5′ ends of nascent RNA polymerase II transcripts is the first pre-mRNA processing event in all eukaryotic cells. Capping enzyme (CE) is recruited to transcription complexes soon after initiation by the phosphorylation of Ser-5 of the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Here, we analyze the role of CE in promoter clearance and its functional interactions with different factors that are involved in promoter clearance. FCP1-mediated dephosphorylation of the carboxyl-terminal domain results in a drastic decrease in cotranscriptional capping efficiency but is reversed by the presence of DRB sensitivity-inducing factor (DSIF). These results suggest involvement of DSIF in CE recruitment. Importantly, CE relieves transcriptional repression by the negative elongation factor, indicating a critical role of CE in the elongation checkpoint control mechanism during promoter clearance. This functional interaction between CE and the negative elongation factor documents a dynamic role of CE in promoter clearance beyond its catalytic activities.

Thermodynamics of ligand binding by the yeast mRNA-capping enzyme reveals different modes of binding

Bougie, Isabelle; Parent, Amélie; Bisaillon, Martin
Fonte: Portland Press Ltd. Publicador: Portland Press Ltd.
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
36.62%
RNA-capping enzymes are involved in the synthesis of the cap structure found at the 5′-end of eukaryotic mRNAs. The present study reports a detailed study on the thermodynamic parameters involved in the interaction of an RNA-capping enzyme with its ligands. Analysis of the interaction of the Saccharomyces cerevisiae RNA-capping enzyme (Ceg1) with GTP, RNA and manganese ions revealed significant differences between the binding forces that drive the interaction of the enzyme with its RNA and GTP substrates. Our thermodynamic analyses indicate that the initial association of GTP with the Ceg1 protein is driven by a favourable enthalpy change (ΔH=−80.9 kJ/mol), but is also clearly associated with an unfavourable entropy change (TΔS=−62.9 kJ/mol). However, the interaction between Ceg1 and RNA revealed a completely different mode of binding, where binding to RNA is clearly dominated by a favourable entropic effect (TΔS=20.5 kJ/mol), with a minor contribution from a favourable enthalpy change (ΔH=−5.3 kJ/mol). Fluorescence spectroscopy also allowed us to evaluate the initial binding of GTP to such an enzyme, thereby separating the GTP binding step from the concomitant metal-dependent hydrolysis of GTP that results in the formation of a covalent GMP–protein intermediate. In addition to the determination of the energetics of ligand binding...

Vesicular Stomatitis Virus mRNA Capping Machinery Requires Specific cis-Acting Signals in the RNA▿

Wang, Jennifer T.; McElvain, Lauren E.; Whelan, Sean P. J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
36.56%
Many viruses of eukaryotes that use mRNA cap-dependent translation strategies have evolved alternate mechanisms to generate the mRNA cap compared to their hosts. The most divergent of these mechanisms are those used by nonsegmented negative-sense (NNS) RNA viruses, which evolved a capping enzyme that transfers RNA onto GDP, rather than GMP onto the 5′ end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we show that mRNA cap formation is further distinct, requiring a specific cis-acting signal in the RNA. Using recombinant VSV, we determined the function of the eight conserved positions of the gene-start sequence in mRNA initiation and cap formation. Alterations to this sequence compromised mRNA initiation and separately formation of the GpppA cap structure. These studies provide genetic and biochemical evidence that the mRNA capping apparatus of VSV evolved an RNA capping machinery that functions in a sequence-specific manner.

The Carboxyl-terminal Domain of RNA Polymerase II Is Not Sufficient to Enhance the Efficiency of Pre-mRNA Capping or Splicing in the Context of a Different Polymerase*S⃞

Natalizio, Barbara J.; Robson-Dixon, Nicole D.; Garcia-Blanco, Mariano A.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 27/03/2009 EN
Relevância na Pesquisa
36.6%
Eukaryotic messenger RNA precursors (pre-mRNAs) synthesized by RNA polymerase II (RNAP II) are processed co-transcriptionally. The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II is thought to mediate the coupling of transcription with pre-mRNA processing by coordinating the recruitment of processing factors during synthesis of nascent transcripts. Previous studies have demonstrated that the phosphorylated CTD is required for efficient co-transcriptional processing. In the study presented here we investigated whether the CTD is sufficient to coordinate transcription with pre-mRNA capping and splicing in the context of two other DNA-dependent RNA polymerases, mammalian RNAP III and bacteriophage T7 RNAP. Our results indicate that the CTD fused to the largest subunit of RNAP III (POLR3A) is not sufficient to enhance co-transcriptional pre-mRNA splicing or capping in vivo. Additionally, we analyzed a T7 RNAP-CTD fusion protein and examined its ability to enhance pre-mRNA splicing and capping of both constitutively and alternatively spliced substrates. We observed that the CTD in the context of T7 RNAP was not sufficient to enhance pre-mRNA splicing or capping either in vitro or in vivo. We propose that the efficient coupling of transcription to pre-mRNA processing requires not only the phosphorylated CTD but also other RNAP II specific subunits or associated factors.

Identification of a Cytoplasmic Complex That Adds a Cap onto 5′-Monophosphate RNA ▿ §

Otsuka, Yuichi; Kedersha, Nancy L.; Schoenberg, Daniel R.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
36.55%
Endonuclease decay of nonsense-containing β-globin mRNA in erythroid cells generates 5′-truncated products that were reported previously to have a cap or caplike structure. We confirmed that this 5′ modification is indistinguishable from the cap on full-length mRNA, and Western blotting, immunoprecipitation, and active-site labeling identified a population of capping enzymes in the cytoplasm of erythroid and nonerythroid cells. Cytoplasmic capping enzyme sediments in a 140-kDa complex that contains a kinase which, together with capping enzyme, converts 5′-monophosphate RNA into 5′-GpppX RNA. Capping enzyme shows diffuse and punctate staining throughout the cytoplasm, and its staining does not overlap with P bodies or stress granules. Expression of inactive capping enzyme in a form that is restricted to the cytoplasm reduced the ability of cells to recover from oxidative stress, thus supporting a role for capping in the cytoplasm and suggesting that some mRNAs may be stored in an uncapped state.

Evidence for a capping enzyme with specificity for the trypanosome spliced leader RNA

Ruan, Jia-peng; Shen, Shuiyuan; Ullu, Elisabetta; Tschudi, Christian
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
36.5%
Capping of the pre-mRNA 5′ end by addition a monomethylated guanosine cap (m7G) is an essential and the earliest modification in the biogenesis of mRNA. The reaction is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. Whereas this modification occurs co-transcriptionally in most eukaryotic organisms, trypanosomatid protozoa mRNAs acquire the m7G cap by trans-splicing, which entails the transfer of the capped spliced leader (SL) from the SL RNA to the mRNA. Intriguingly, the genomes of all trypanosomatid protozoa sequenced to date possess two distinct proteins with the signature motifs of guanylyltransferases: TbCGM1 and the previously characterized TbCE1. Here we provide biochemical evidence that TbCgm1 is a capping enzyme. Whereas RNAi-induced downregulation of TbCe1 had no phenotypic consequences, we found that TbCGM1 is essential for trypanosome viability and is required for SL RNA capping. Furthermore, consistent with co-transcriptional addition of the m7G cap, chromatin immunoprecipitation revealed recruitment of TbCgm1 to the SL RNA genes.

A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

Suh, Man-Hee; Meyer, Peter A.; Gu, Meigang; Ye, Ping; Zhang, Mincheng; Kaplan, Craig D.; Lima, Christopher D.; Fu, Jianhua
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
46.43%
RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5′-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes.

Analysis of RNA Binding by the Dengue Virus NS5 RNA Capping Enzyme

Henderson, Brittney R.; Saeedi, Bejan J.; Campagnola, Grace; Geiss, Brian J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 12/10/2011 EN
Relevância na Pesquisa
46.72%
Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5′ end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the KD for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5′ phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA...

The Immunosuppressive Agent Mizoribine Monophosphate Is an Inhibitor of the Human RNA Capping Enzyme

Picard-Jean, Frédéric; Bougie, Isabelle; Shuto, Satoshi; Bisaillon, Martin
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 17/01/2013 EN
Relevância na Pesquisa
46.58%
Mizoribine monophosphate (MZP) is a specific inhibitor of the cellular inosine-5′-monophosphate dehydrogenase (IMPDH), the enzyme catalyzing the rate-limiting step of de novo guanine nucleotide biosynthesis. MZP is a highly potent antagonistic inhibitor of IMPDH that blocks the proliferation of T and B lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. In the present study, we investigated the ability of MZP to directly inhibit the human RNA capping enzyme (HCE), a protein harboring both RNA 5′-triphosphatase and RNA guanylyltransferase activities. HCE is involved in the synthesis of the cap structure found at the 5′ end of eukaryotic mRNAs, which is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation of mRNAs. Our biochemical studies provide the first insight that MZP can inhibit the formation of the RNA cap structure catalyzed by HCE. In the presence of MZP, the RNA 5′-triphosphatase activity appears to be relatively unaffected while the RNA guanylyltransferase activity is inhibited, indicating that the RNA guanylyltransferase activity is the main target of MZP inhibition. Kinetic studies reveal that MZP is a non-competitive inhibitor that likely targets an allosteric site on HCE. Mizoribine also impairs mRNA capping in living cells...

Enzymatic Synthesis of RNAs Capped with Nucleotide Analogues Reveals the Molecular Basis for Substrate Selectivity of RNA Capping Enzyme: Impacts on RNA Metabolism

Issur, Moheshwarnath; Bougie, Isabelle; Despins, Simon; Bisaillon, Martin
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 25/09/2013 EN
Relevância na Pesquisa
46.7%
RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5’ ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Parameciumbursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs...

Regulation of Flavivirus RNA Synthesis and Capping

Saeedi, Bejan J.; Geiss, Brian J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
36.5%
RNA viruses, such as flaviviruses, are able to efficiently replicate and cap their RNA genomes in vertebrate and invertebrate cells. Flaviviruses use several specialized proteins to first make an uncapped negative strand copy of the viral genome that is used as a template for the synthesis of large numbers of capped genomic RNAs. Despite using relatively simple mechanisms to replicate their RNA genomes, there are significant gaps in our understanding of how flaviviruses switch between negative and positive strand RNA synthesis and how RNA capping is regulated. Recent work has begun to provide a conceptual framework for flavivirus RNA replication and capping and shown some surprising roles for genomic RNA during replication and pathogenesis.

Flaviviral Replication Complex: Coordination between RNA Synthesis and 5′-RNA Capping

Klema, Valerie J.; Padmanabhan, Radhakrishnan; Choi, Kyung H.
Fonte: MDPI Publicador: MDPI
Tipo: Artigo de Revista Científica
Publicado em 13/08/2015 EN
Relevância na Pesquisa
46.5%
Genome replication in flavivirus requires (−) strand RNA synthesis, (+) strand RNA synthesis, and 5′-RNA capping and methylation. To carry out viral genome replication, flavivirus assembles a replication complex, consisting of both viral and host proteins, on the cytoplasmic side of the endoplasmic reticulum (ER) membrane. Two major components of the replication complex are the viral non-structural (NS) proteins NS3 and NS5. Together they possess all the enzymatic activities required for genome replication, yet how these activities are coordinated during genome replication is not clear. We provide an overview of the flaviviral genome replication process, the membrane-bound replication complex, and recent crystal structures of full-length NS5. We propose a model of how NS3 and NS5 coordinate their activities in the individual steps of (−) RNA synthesis, (+) RNA synthesis, and 5′-RNA capping and methylation.

Bromovirus RNA Replication Compartment Formation Requires Concerted Action of 1a's Self-Interacting RNA Capping and Helicase Domains

Diaz, Arturo; Gallei, Andreas; Ahlquist, Paul
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2012 EN
Relevância na Pesquisa
46.73%
All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular...

Conversion of a DNA ligase into an RNA capping enzyme.

Doherty, A J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/08/1999 EN
Relevância na Pesquisa
46.59%
In eukaryotes, newly synthesised mRNA is 'capped' by the addition of GMP to the 5" end by RNA capping enzymes. Recent structural studies have shown that RNA capping enzymes and DNA ligases have similar protein folds, suggesting a conserved catalytic mechanism. To explore these similarities we have produced a chimeric enzyme comprising the N-terminal domain 1 of a DNA ligase fused to the C-terminal domain 2 of a mRNA capping enzyme. This report shows that this hybrid enzyme retains adenylation activity, characteristic of DNA ligases but, remarkably, the chimera has ATP-dependent mRNA capping activity. This is the first observation of ATP-dependent RNA capping. These results suggest that nucleotidyltransferases may have evolved from a common ancestral gene.

Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA.

Cong, P; Shuman, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1995 EN
Relevância na Pesquisa
36.68%
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...

Expression and characterization of an RNA capping enzyme encoded by Chlorella virus PBCV-1.

Ho, C K; Van Etten, J L; Shuman, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1996 EN
Relevância na Pesquisa
36.46%
We report that the A103R protein of Chlorella virus PBCV-1 is an mRNA capping enzyme that catalyzes the transfer of GMP from GTP to the 5' diphosphate end of RNA. This is a two-step reaction in which the enzyme first condenses with GTP to form a covalent enzyme-GMP intermediate and then transfers the GMP to an RNA acceptor to form a GpppN cap. Purified recombinant Al03R is a 38-kDa monomer that lacks RNA (guanine-7-) methyltransferase activity. With respect to its size, amino acid sequence, and biochemical properties, A103R is more closely related to the yeast RNA guanylyltransferases than it is to the multifunctional capping enzymes coded for by other large DNA viruses--the poxviruses and African swine fever virus. We surmise that in order to cap its transcripts, PBCV-l must either encode additional 5' processing activities or else rely on the host alga to provide these functions.

Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases.

Shuman, S; Liu, Y; Schwer, B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 06/12/1994 EN
Relevância na Pesquisa
36.53%
Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably...