The Experts below are selected from a list of 1662 Experts worldwide ranked by ideXlab platform
Jon S. Thorson - One of the best experts on this subject based on the ideXlab platform.
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a comparison of sugar indicators enables a universal high throughput sugar 1 phosphate Nucleotidyltransferase assay
Analytical Biochemistry, 2008Co-Authors: Rocco Moretti, Jon S. ThorsonAbstract:A systematic comparison of six sugar indicators for their sensitivity, specificity, cross-reactivity, and suitability in the context of crude lysates revealed para-hydroxybenzoic acid hydrazide (pHBH) to be best suited for application in a plate-based phosphatase-assisted universal sugar-1-phosphate Nucleotidyltransferase assay. The addition of a general phosphatase to Nucleotidyltransferase reaction aliquots enabled the conversion of remaining sugar-1-phosphate to free sugar, the concentration of which could be rapidly assessed via the pHBH assay. The assay was validated using the model glucose-1-phosphate thymidylyltransferase from Salmonella enterica (RmlA) and compared favorably with a previously reported HPLC assay. This coupled discontinuous assay is quantitative, high throughput, and robust; relies only on commercially available enzymes and reagents; does not require chromatography, specialized detectors (e.g., mass or evaporative light scattering detectors), or radioisotopes; and is capable of detecting less than 5 nmol of sugar-1-phosphate. It is anticipated that this high-throughput assay system will greatly facilitate Nucleotidyltransferase mechanistic and directed evolution/engineering studies.
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enhancing the latent nucleotide triphosphate flexibility of the glucose 1 phosphate thymidylyltransferase rmla
Journal of Biological Chemistry, 2007Co-Authors: Rocco Moretti, Jon S. ThorsonAbstract:Nucleotidyltransferases are central to nearly all glycosylation-dependent processes and have been used extensively for the chemoenzymatic synthesis of sugar nucleotides. The determination of the NTP specificity of the model thymidylyltransferase RmlA revealed RmlA to utilize all eight naturally occurring NTPs with varying levels of catalytic efficiency, even in the presence of nonnative sugar-1-phosphates. Guided by structural models, active site engineering of RmlA led to alterations of the inherent pyrimidine/purine bias by up to three orders of magnitude. This study sets the stage for engineering single universal Nucleotidyltransferases and also provides new catalysts for the synthesis of novel nucleotide diphosphosugars.
Mario Morl - One of the best experts on this subject based on the ideXlab platform.
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cca addition gone wild unusual occurrence and phylogeny of four different trna Nucleotidyltransferases in acanthamoeba castellanii
Molecular Biology and Evolution, 2021Co-Authors: Lieselotte Erber, Heike Betat, Mario MorlAbstract:tRNAs are important players in the protein synthesis machinery, where they act as adapter molecules for translating the mRNA codons into the corresponding amino acid sequence. In a series of highly conserved maturation steps, the primary transcripts are converted into mature tRNAs. In the amoebozoan Acanthamoeba castellanii, a highly unusual evolution of some of these processing steps was identified that are based on unconventional RNA polymerase activities. In this context, we investigated the synthesis of the 3'-terminal CCA-end that is added posttranscriptionally by a specialized polymerase, the tRNA Nucleotidyltransferase (CCA-adding enzyme). The majority of eukaryotic organisms carry only a single gene for a CCA-adding enzyme that acts on both the cytosolic and the mitochondrial tRNA pool. In a bioinformatic analysis of the genome of this organism, we identified a surprising multitude of genes for enzymes that contain the active site signature of eukaryotic/eubacterial tRNA Nucleotidyltransferases. In vitro activity analyses of these enzymes revealed that two proteins represent bona fide CCA-adding enzymes, one of them carrying an N-terminal sequence corresponding to a putative mitochondrial target signal. The other enzymes have restricted activities and represent CC- and A-adding enzymes, respectively. The A-adding enzyme is of particular interest, as its sequence is closely related to corresponding enzymes from Proteobacteria, indicating a horizontal gene transfer. Interestingly, this unusual diversity of Nucleotidyltransferase genes is not restricted to Acanthamoeba castellanii but is also present in other members of the Acanthamoeba genus, indicating an ancient evolutionary trait.
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A Temporal Order in 5′- and 3′- Processing of Eukaryotic tRNAHis
'MDPI AG', 2019Co-Authors: Marie-theres Pöhler, Heike Betat, Tracy M. Roach, Jane E. Jackman, Mario MorlAbstract:For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3′-terminal CCA sequence that includes the site of aminoacylation, the additional 5′-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3′-CCA and 5′-G-1 are added post-transcriptionally by tRNA Nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA Nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3′-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition
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the ancestor of modern holozoa acquired the cca adding enzyme from alphaproteobacteria by horizontal gene transfer
Nucleic Acids Research, 2015Co-Authors: Heike Betat, Tobias Mede, Sandy Tretbar, Lydia Steiner, Peter F Stadler, Mario Morl, Sonja J ProhaskaAbstract:Transfer RNAs (tRNAs) require the absolutely conserved sequence motif CCA at their 3′-ends, representing the site of aminoacylation. In the majority of organisms, this trinucleotide sequence is not encoded in the genome and thus has to be added post-transcriptionally by the CCA-adding enzyme, a specialized Nucleotidyltransferase. In eukaryotic genomes this ubiquitous and highly conserved enzyme family is usually represented by a single gene copy. Analysis of published sequence data allows us to pin down the unusual evolution of eukaryotic CCA-adding enzymes. We show that the CCA-adding enzymes of animals originated from a horizontal gene transfer event in the stem lineage of Holozoa, i.e. Metazoa (animals) and their unicellular relatives, the Choanozoa. The tRNA Nucleotidyltransferase, acquired from an α-proteobacterium, replaced the ancestral enzyme in Metazoa. However, in Choanoflagellata, the group of Choanozoa that is closest to Metazoa, both the ancestral and the horizontally transferred CCA-adding enzymes have survived. Furthermore, our data refute a mitochondrial origin of the animal tRNA Nucleotidyltransferases.
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tRNA Nucleotidyltransferases: ancient catalysts with an unusual mechanism of polymerization
Cellular and Molecular Life Sciences, 2010Co-Authors: Heike Betat, Christiane Rammelt, Mario MorlAbstract:RNA polymerases are important enzymes involved in the realization of the genetic information encoded in the genome. Thereby, DNA sequences are used as templates to synthesize all types of RNA. Besides these classical polymerases, there exists another group of RNA polymerizing enzymes that do not depend on nucleic acid templates. Among those, tRNA Nucleotidyltransferases show remarkable and unique features. These enzymes add the nucleotide triplet C–C–A to the 3′-end of tRNAs at an astonishing fidelity and are described as “CCA-adding enzymes”. During this incorporation of exactly three nucleotides, the enzymes have to switch from CTP to ATP specificity. How these tasks are fulfilled by rather simple and small enzymes without the help of a nucleic acid template is a fascinating research area. Surprising results of biochemical and structural studies allow scientists to understand at least some of the mechanistic principles of the unique polymerization mode of these highly unusual enzymes.
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trna Nucleotidyltransferases highly unusual rna polymerases with vital functions
FEBS Letters, 2010Co-Authors: Stefan Vortler, Mario MorlAbstract:tRNA-Nucleotidyltransferases are fascinating and unusual RNA polymerases responsible for the synthesis of the nucleotide triplet CCA at the 3'-terminus of tRNAs. As this CCA end represents an essential functional element for aminoacylation and translation, these polymerases (CCA-adding enzymes) are of vital importance in all organisms. With a possible origin of ancient telomerase-like activity, the CCA-adding enzymes obviously emerged twice during evolution, leading to structurally different, but functionally identical enzymes. The evolution as well as the unique polymerization features of these interesting proteins will be discussed in this review.
Rocco Moretti - One of the best experts on this subject based on the ideXlab platform.
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a comparison of sugar indicators enables a universal high throughput sugar 1 phosphate Nucleotidyltransferase assay
Analytical Biochemistry, 2008Co-Authors: Rocco Moretti, Jon S. ThorsonAbstract:A systematic comparison of six sugar indicators for their sensitivity, specificity, cross-reactivity, and suitability in the context of crude lysates revealed para-hydroxybenzoic acid hydrazide (pHBH) to be best suited for application in a plate-based phosphatase-assisted universal sugar-1-phosphate Nucleotidyltransferase assay. The addition of a general phosphatase to Nucleotidyltransferase reaction aliquots enabled the conversion of remaining sugar-1-phosphate to free sugar, the concentration of which could be rapidly assessed via the pHBH assay. The assay was validated using the model glucose-1-phosphate thymidylyltransferase from Salmonella enterica (RmlA) and compared favorably with a previously reported HPLC assay. This coupled discontinuous assay is quantitative, high throughput, and robust; relies only on commercially available enzymes and reagents; does not require chromatography, specialized detectors (e.g., mass or evaporative light scattering detectors), or radioisotopes; and is capable of detecting less than 5 nmol of sugar-1-phosphate. It is anticipated that this high-throughput assay system will greatly facilitate Nucleotidyltransferase mechanistic and directed evolution/engineering studies.
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enhancing the latent nucleotide triphosphate flexibility of the glucose 1 phosphate thymidylyltransferase rmla
Journal of Biological Chemistry, 2007Co-Authors: Rocco Moretti, Jon S. ThorsonAbstract:Nucleotidyltransferases are central to nearly all glycosylation-dependent processes and have been used extensively for the chemoenzymatic synthesis of sugar nucleotides. The determination of the NTP specificity of the model thymidylyltransferase RmlA revealed RmlA to utilize all eight naturally occurring NTPs with varying levels of catalytic efficiency, even in the presence of nonnative sugar-1-phosphates. Guided by structural models, active site engineering of RmlA led to alterations of the inherent pyrimidine/purine bias by up to three orders of magnitude. This study sets the stage for engineering single universal Nucleotidyltransferases and also provides new catalysts for the synthesis of novel nucleotide diphosphosugars.
George H Jones - One of the best experts on this subject based on the ideXlab platform.
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phylogeny and evolution of rna 3 Nucleotidyltransferases in bacteria
Journal of Molecular Evolution, 2019Co-Authors: George H JonesAbstract:The tRNA Nucleotidyltransferases and poly(A) polymerases belong to a superfamily of Nucleotidyltransferases. The amino acid sequences of a number of bacterial tRNA Nucleotidyltransferases and poly(A) polymerases have been used to construct a rooted, neighbor-joining phylogenetic tree. Using information gleaned from that analysis, along with data from the rRNA-based phylogenetic tree, structural data available on a number of members of the superfamily and other biochemical information on the superfamily, it is possible to suggest a scheme for the evolution of the bacterial tRNA Nucleotidyltransferases and poly(A) polymerases from ancestral species. Elements of that scheme are discussed along with questions arising from the scheme which can be explored experimentally.
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geobacter sulfurreducens contains separate c and a adding trna Nucleotidyltransferases and a poly a polymerase
Journal of Bacteriology, 2009Co-Authors: Patricia Bralley, Madeline Cozad, George H JonesAbstract:The genome of Geobacter sulfurreducens contains three genes whose sequences are quite similar to sequences encoding known members of an RNA Nucleotidyltransferase superfamily that includes tRNA Nucleotidyltransferases and poly(A) polymerases. Reverse transcription-PCR using G. sulfurreducens total RNA demonstrated that the genes encoding these three proteins are transcribed. These genes, encoding proteins designated NTSFI, NTSFII, and NTSFIII, were cloned and overexpressed in Escherichia coli. The corresponding enzymes were purified and assayed biochemically, resulting in identification of NTSFI as a poly(A) polymerase, NTSFII as a C-adding tRNA Nucleotidyltransferase, and NTSFIII as an A-adding tRNA Nucleotidyltransferase. Analysis of G. sulfurreducens rRNAs and mRNAs revealed the presence of heteropolymeric RNA 3′ tails. This is the first characterization of a bacterial system that expresses separate C- and A-adding tRNA Nucleotidyltransferases and a poly(A) polymerase.
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a phylogeny of bacterial rna Nucleotidyltransferases bacillus halodurans contains two trna Nucleotidyltransferases
Journal of Bacteriology, 2005Co-Authors: Patricia Bralley, Samantha A Chang, George H JonesAbstract:We have analyzed the distribution of RNA Nucleotidyltransferases from the family that includes poly(A) polymerases (PAP) and tRNA Nucleotidyltransferases (TNT) in 43 bacterial species. Genes of several bacterial species encode only one member of the Nucleotidyltransferase superfamily (NTSF), and if that protein functions as a TNT, those organisms may not contain a poly(A) polymerase I like that of Escherichia coli. The genomes of several of the species examined encode more than one member of the Nucleotidyltransferase superfamily. The function of some of those proteins is known, but in most cases no biochemical activity has been assigned to the NTSF. The NTSF protein sequences were used to construct an unrooted phylogenetic tree. To learn more about the function of the NTSFs in species whose genomes encode more than one, we have examined Bacillus halodurans. We have demonstrated that B. halodurans adds poly(A) tails to the 3' ends of RNAs in vivo. We have shown that the genes for both of the NTSFs encoded by the B. halodurans genome are transcribed in vivo. We have cloned, overexpressed, and purified the two NTSFs and have shown that neither functions as poly(A) polymerase in vitro. Rather, the two proteins function as tRNA Nucleotidyltransferases, and our data suggest that, like some of the deep branching bacterial species previously studied by others, B. halodurans possesses separate CC- and A-adding tRNA Nucleotidyltransferases. These observations raise the interesting question of the identity of the enzyme responsible for RNA polyadenylation in Bacillus.
Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus
Structure, 2014Co-Authors: O.j.p. Kyrieleis, Stewart Shuman, Jonathan Chang, Marcos De La Peña, Stephen CusackAbstract:Summary Vaccinia virus capping enzyme is a heterodimer of D1 (844 aa) and D12 (287 aa) polypeptides that executes all three steps in m 7 GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA triphosphatase (TPase)-guanylyltransferase (GTase) module and a C-terminal guanine-N7-methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1⋅D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloenzyme and covalent Nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase Nucleotidyltransferase and OB-fold domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1⋅D12 structure complements and rationalizes four decades of biochemical studies of this enzyme, which was the first capping enzyme to be purified and characterized, and provides new insights into the origins of the capping systems of other large DNA viruses.
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sequence specific 1hn 13c and 15n backbone resonance assignments of the 34 kda paramecium bursaria chlorella virus 1 pbcv1 dna ligase
Biomolecular Nmr Assignments, 2009Co-Authors: Andrea Piserchio, Stewart Shuman, Pravin A Nair, Ranajeet GhoseAbstract:Chlorella virus DNA ligase (ChVLig) is a minimal (298-amino acid) pluripotent ATP-dependent ligase composed of three structural modules—a Nucleotidyltransferase domain, an OB domain, and a β-hairpin latch—that forms a circumferential clamp around nicked DNA. ChVLig provides an instructive model to understand the chemical and conformational steps of nick repair. Here we report the assignment of backbone 13C, 15N, 1HN resonances of this 34.2 kDa protein, the first for a DNA ligase in full-length form.
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role of Nucleotidyltransferase motifs i iii and iv in the catalysis of phosphodiester bond formation by chlorella virus dna ligase
Nucleic Acids Research, 2002Co-Authors: Verl Sriskanda, Stewart ShumanAbstract:ATP-dependent DNA ligases catalyze the sealing of 5'-phosphate and 3'-hydroxyl termini at DNA nicks by means of a series of three nucleotidyl transfer steps. Here we have analyzed by site-directed mutagenesis the roles of conserved amino acids of Chlorella virus DNA ligase during the third step of the ligation pathway, which entails reaction of the 3'-OH of the nick with the DNA-adenylate intermediate to form a phosphodiester and release AMP. We found that Asp65 and Glu67 in Nucleotidyltransferase motif III and Glu161 in motif IV enhance the rate of step 3 phosphodiester formation by factors of 20, 1000 and 60, respectively. Asp29 and Arg32 in Nucleotidyltransferase motif I enhance the rate of step 3 by 60-fold. Gel shift analysis showed that mutations of Arg32 and Asp65 suppressed ligase binding to a pre-adenylated nick, whereas Asp29, Glu67 and Glu161 mutants bound stably to DNA-adenylate. We infer that Asp29, Glu67 and Glu161 are involved directly in the step 3 reaction. In several cases, the effects of alanine or conservative mutations on step 3 were modest compared to their effects on the composite ligation reaction and individual upstream steps. These results, in concert with available crystallographic data, suggest that the active site of DNA ligase is remodeled during the three steps of the pathway and that some of the catalytic side chains play distinct roles at different stages.
