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

2004 ◽  
Vol 384 (2) ◽  
pp. 411-420 ◽  
Author(s):  
Isabelle BOUGIE ◽  
Amélie PARENT ◽  
Martin BISAILLON
Keyword(s):  
Ligand Binding ◽  
Entropy Change ◽  
Enthalpy Change ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Eukaryotic Mrnas ◽  
Entropic Effect ◽  
A Minor ◽  
Rna Capping ◽  
Capping Enzymes

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, our study leads to a better understanding of the molecular basis of substrate recognition by RNA-capping enzymes.

scholarly journals Trypanosome Capping Enzymes Display a Novel Two-Domain Structure

1998 ◽  
Vol 18 (8) ◽  
pp. 4612-4619 ◽  
Author(s):  
Erika Silva ◽  
Elisabetta Ullu ◽  
Ryuji Kobayashi ◽  
Christian Tschudi
Keyword(s):  
Domain Structure ◽  
Consensus Sequence ◽  
Rna Modification ◽  
Phosphate Binding ◽  
Amino Terminal ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Eukaryotic Mrnas ◽  
Carboxy Terminal ◽  
Capping Enzymes

ABSTRACT The ubiquitous m7G cap of eukaryotic mRNAs and of precursors to the spliceosomal small nuclear RNAs (snRNAs) is the result of an essential RNA modification acquired during transcript elongation. In trypanosomes, the m7G cap is restricted to the spliced leader (SL) RNA and the precursors of U2, U3, and U4 snRNAs. mRNA capping in these organisms occurs posttranscriptionally bytrans splicing, which transfers the capped SL sequence to the 5′ ends of all mRNAs. The SL cap is the most elaborate cap structure known in nature and has been shown to consist of an m7G residue followed by four methylated nucleotides. UsingCrithidia fasciculata, we have characterized and purified the guanylyltransferase (capping enzyme), which transfers GMP from GTP to the diphosphate end of RNA. The corresponding gene codes for a protein of 697 amino acids, with the carboxy-terminal half of theC. fasciculata guanylyltransferase containing the six signature motifs previously identified in yeast capping enzymes. The amino-terminal half contains a domain that displays no resemblance to any other domain associated with capping enzymes. Intriguingly, this region harbors a consensus sequence for a phosphate-binding loop which is found in ATP- and GTP-binding proteins. This two-domain structure is also present in the Trypanosoma brucei capping enzyme, which shows 44% overall identity with the C. fasciculatacapping enzyme. Thus, this structure appears to be common to all trypanosomatid protozoa and defines a novel class of capping enzymes.

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

1995 ◽  
Vol 15 (11) ◽  
pp. 6222-6231 ◽  
Author(s):  
P Cong ◽  
S Shuman
Keyword(s):  
Vaccinia Virus ◽  
Active Sites ◽  
Mutational Analysis ◽  
Structural Basis ◽  
Sequence Element ◽  
Termination Factor ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Rna Capping ◽  
Capping Enzymes

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.

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

2007 ◽  
Vol 81 (20) ◽  
pp. 11499-11506 ◽  
Author(s):  
Jennifer T. Wang ◽  
Lauren E. McElvain ◽  
Sean P. J. Whelan
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Translation Strategies ◽  
Cis Acting ◽  
Specific Manner ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Vesicular Stomatitis ◽  
Rna Capping ◽  
Negative Sense

ABSTRACT 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.

scholarly journals Deletion of Individual mRNA Capping Genes Is Unexpectedly Not Lethal to Candida albicans and Results in Modified mRNA Cap Structures

2002 ◽  
Vol 1 (6) ◽  
pp. 1010-1020 ◽  
Author(s):  
Donna S. Dunyak ◽  
Daniel S. Everdeen ◽  
Joseph G. Albanese ◽  
Cheryl L. Quinn
Keyword(s):  
Candida Albicans ◽  
Molecular Targets ◽  
Alternative Pathways ◽  
Redundant Genes ◽  
Mrna Capping ◽  
Genes Encoding ◽  
Eukaryotic Mrnas ◽  
Encoding Gene ◽  
Capping Enzymes ◽  
Null Mutants

ABSTRACT Eukaryotic mRNAs are modified at the 5′ end with a cap structure. In fungal cells, the formation of the mRNA cap structure is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and methyltransferase. Fungal capping enzymes have been proposed to be good antifungal targets because they differ significantly from their human counterparts and the genes encoding these enzymes are essential in Saccharomyces cerevisiae. In the present study, Candida albicans null mutants were constructed for both the mRNA triphosphatase-encoding gene (CET1) and the mRNA methyltransferase encoding gene (CCM1), proving that these genes are not essential in C. albicans. Heterozygous deletions were generated, but no null mutants were isolated for the guanylyltransferase-encoding gene (CGT1), indicating that this gene probably is essential in C. albicans. Whereas these results indicate that Cet1p and Ccm1p are not ideal molecular targets for development of anticandidal drugs, they do raise questions about the capping of mRNA and translation initiation in this fungus. Southern blot analysis of genomic DNA indicates that there are not redundant genes for CET1 and CCM1 and analysis of mRNA cap structures indicate there are not alternative pathways compensating for the function of CET1 or CCM1 in the null mutants. Instead, it appears that C. albicans can survive with modified mRNA cap structures.

scholarly journals Identification of a Cytoplasmic Complex That Adds a Cap onto 5′-Monophosphate RNA

2009 ◽  
Vol 29 (8) ◽  
pp. 2155-2167 ◽  
Author(s):  
Yuichi Otsuka ◽  
Nancy L. Kedersha ◽  
Daniel R. Schoenberg
Keyword(s):  
Oxidative Stress ◽  
Western Blotting ◽  
Active Site ◽  
Globin Mrna ◽  
P Bodies ◽  
Capping Enzyme ◽  
Rna Capping ◽  
Capping Enzymes ◽  
Punctate Staining ◽  
Cytoplasmic Complex

ABSTRACT 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.

scholarly journals Yeast-Based Genetic System for Functional Analysis of Poxvirus mRNA Cap Methyltransferase

2003 ◽  
Vol 77 (13) ◽  
pp. 7300-7307 ◽  
Author(s):  
Nayanendu Saha ◽  
Stewart Shuman ◽  
Beate Schwer
Keyword(s):  
Vaccinia Virus ◽  
Genetic Model ◽  
Genetic System ◽  
In Vivo Studies ◽  
Methyltransferase Activity ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Capping Enzymes

ABSTRACT Structural differences between poxvirus and human mRNA capping enzymes recommend cap formation as a target for antipoxviral drug discovery. Genetic and pharmacologic analysis of the poxvirus capping enzymes requires in vivo assays in which the readout depends on the capacity of the viral enzyme to catalyze cap synthesis. Here we have used the budding yeast Saccharomyces cerevisiae as a genetic model for the study of poxvirus cap guanine-N7 methyltransferase. The S. cerevisiae capping system consists of separate triphosphatase (Cet1), guanylyltransferase (Ceg1), and methyltransferase (Abd1) components. All three activities are essential for cell growth. We report that the methyltransferase domain of vaccinia virus capping enzyme (composed of catalytic vD1-C and stimulatory vD12 subunits) can function in lieu of yeast Abd1. Coexpression of both vaccinia virus subunits is required for complementation of the growth of abd1Δ cells. Previously described mutations of vD1-C and vD12 that eliminate or reduce methyltransferase activity in vitro either abolish abd1Δ complementation or elicit conditional growth defects. We have used the yeast complementation assay as the primary screen in a new round of alanine scanning of the catalytic subunit. We thereby identified several new amino acids that are critical for cap methylation activity in vivo. Studies of recombinant proteins show that the lethal vD1-C mutations do not preclude heterodimerization with vD12 but either eliminate or reduce cap methyltransferase activity in vitro.

scholarly journals A Yeast-Based Genetic System for Functional Analysis of Viral mRNA Capping Enzymes

2000 ◽  
Vol 74 (12) ◽  
pp. 5486-5494 ◽  
Author(s):  
C. Kiong Ho ◽  
Alexandra Martins ◽  
Stewart Shuman
Keyword(s):  
Fusion Protein ◽  
Vaccinia Virus ◽  
Rna Polymerase Ii ◽  
Elongation Complex ◽  
Mrna Capping ◽  
Rna Triphosphatase ◽  
Capping Enzyme ◽  
Capping Enzymes

ABSTRACT Virus-encoded mRNA capping enzymes are attractive targets for antiviral therapy, but functional studies have been limited by the lack of genetically tractable in vivo systems that focus exclusively on the RNA-processing activities of the viral proteins. Here we have developed such a system by engineering a viral capping enzyme—vaccinia virus D1(1-545)p, an RNA triphosphatase and RNA guanylyltransferase—to function in the budding yeast Saccharomyces cerevisiae in lieu of the endogenous fungal triphosphatase (Cet1p) and guanylyltransferase (Ceg1p). This was accomplished by fusion of D1(1-545)p to the C-terminal guanylyltransferase domain of mammalian capping enzyme, Mce1(211-597)p, which serves as a vehicle to target the viral capping enzyme to the RNA polymerase II elongation complex. An inactivating mutation (K294A) of the mammalian guanylyltransferase active site in the fusion protein had no impact on genetic complementation of cet1Δceg1Δ cells, thus proving that (i) the viral guanylyltransferase was active in vivo and (ii) the mammalian domain can serve purely as a chaperone to direct other proteins to the transcription complex. Alanine scanning had identified five amino acids of vaccinia virus capping enzyme—Glu37, Glu39, Arg77, Glu192, and Glu194—that are essential for γ phosphate cleavage in vitro. Here we show that the introduction of mutation E37A, R77A, or E192A into the fusion protein abrogates RNA triphosphatase function in vivo. The essential residues are located within three motifs that define a family of viral and fungal metal-dependent phosphohydrolases with a distinctive capacity to hydrolyze nucleoside triphosphates to nucleoside diphosphates in the presence of manganese or cobalt. The acidic residues Glu37, Glu39, and Glu192 likely comprise the metal-binding site of vaccinia virus triphosphatase, insofar as their replacement by glutamine abolishes the RNA triphosphatase and ATPase activities.

scholarly journals Critical Residues for GTP Methylation and Formation of the Covalent m7GMP-Enzyme Intermediate in the Capping Enzyme Domain of Bamboo Mosaic Virus

2004 ◽  
Vol 78 (3) ◽  
pp. 1271-1280 ◽  
Author(s):  
Yih-Leh Huang ◽  
Yu-Tsung Han ◽  
Ya-Ting Chang ◽  
Yau-Heiu Hsu ◽  
Menghsiao Meng
Keyword(s):  
Amino Acids ◽  
Mosaic Virus ◽  
Fold Increase ◽  
Methyltransferase Activity ◽  
Reading Frame ◽  
Chromatography Analysis ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Capping Enzymes ◽  
Enzyme Intermediate

ABSTRACT Open reading frame 1 of Bamboo mosaic virus (BaMV), a Potexvirus in the alphavirus-like superfamily, encodes a 155-kDa replicase responsible for the formation of the 5′ cap structure and replication of the viral RNA genome. The N-terminal domain of the viral replicase functions as an mRNA capping enzyme, which exhibits both GTP methyltransferase and S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activities. We mutated each of the four conserved amino acids among the capping enzymes of members within alphavirus-like superfamily and a dozen of other residues to gain insight into the structure-function relationship of the viral enzyme. The mutant enzymes were purified and subsequently characterized. H68A, the mutant enzyme bearing a substitution at the conserved histidine residue, has an ∼10-fold increase in GTP methyltransferase activity but completely loses the ability to form the covalent m7GMP-enzyme intermediate. High-pressure liquid chromatography analysis confirmed the production of m7GTP by the GTP methyltransferase activity of H68A. Furthermore, the produced m7GTP sustained the formation of the m7GMP-enzyme intermediate for the wild-type enzyme in the presence of S-adenosylhomocysteine (AdoHcy), suggesting that the previously observed AdoMet-dependent guanylation of the enzyme using GTP results from reactions of GTP methylation and subsequently guanylation of the enzyme using m7GTP. Mutations occurred at the other three conserved residues (D122, R125, and Y213), and H66 resulted in abolition of activities for both GTP methylation and formation of the covalent m7GMP-enzyme intermediate. Mutations of amino acids such as K121, C234, D310, W312, R316, K344, W406, and K409 decreased both activities by various degrees, and the extents of mutational effects follow similar trends. The affinity to AdoMet of the various BaMV capping enzymes, except H68A, was found in good correlations with not only the magnitude of GTP methyltransferase activity but also the capability of forming the m7GMP-enzyme intermediate. Taken together with the AdoHcy dependence of guanylation of the enzyme using m7GTP, a basic working mechanism, with the contents of critical roles played by the binding of AdoMet/AdoHcy, of the BaMV capping enzyme is proposed and discussed.

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