tRNA Phe Research Articles

TRNA Arg binds in vitro TDP-43 RNA recognition motifs and ligand of Ate1 protein LIAT1.

Transactive response DNA-binding protein 43 (TDP-43) is important for RNA metabolism in all animals and its malfunctions are linked to neurodegenerative and myodegenerative diseases in humans. Arginyl transferase Ate1 transfers an arginyl group from arginylated tRNA Arg to proteolytic fragments of the C-terminal region of TDP-43, prompting their degradation by the ubiquitin proteasome system, thus contributing to TDP-43 proteostasis. To gain more insight into the molecular basis of TDP-43 arginylation, we tested if tRNA Arg could bind in vitro to a panel of recombinant multidomain constructs of human TDP-43 or to the arginylation cofactor protein LIAT1. We observed that in vitro- transcribed human tRNA Arg directly interacts with the RNA recognition motifs of TDP-43 and that their binding is stabilized by dimerization, which is promoted by the amino-terminal domain and the nuclear localization signal sequence of TDP-43. Moreover, the same human TDP-43 constructs that bind tRNA Arg bind native fungal tRNA Phe , suggesting that TDP-43 can bind different populations of tRNAs. Interestingly, human tRNA Arg is also able to bind recombinant mouse LIAT1 suggesting, for the first time, that LIAT1 is an RNA-binding protein. Our findings open a new perspective on the intricate crosstalk between protein and tRNA metabolism, which may eventually contribute to the understanding of the role of TDP-43 proteostasis in health and disease.

The Spatiotemporal Dynamics and Microevolution Events That Favored the Success of the Highly Clonal Multidrug-Resistant Monophasic Salmonella Typhimurium Circulating in Europe.

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3′ end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

Duplication and diversification of a unique chromosomal virulence island hosting the subtilase cytotoxin in Escherichia coli ST58.

The AB5 cytotoxins are important virulence factors in Escherichia coli . The most notable members of the AB5 toxin families include Shiga toxin families 1 (Stx1) and 2 (Stx2), which are associated with enterohaemorrhagic E. coli infections causing haemolytic uraemic syndrome and haemorrhagic colitis. The subAB toxins are the newest and least well understood members of the AB5 toxin gene family. The subtilase toxin genes are divided into a plasmid-based variant, subAB1, originally described in enterohaemorrhagic E. coli O113:H21, and distinct chromosomal variants, subAB2, that reside in pathogenicity islands encoding additional virulence effectors. Previously we identified a chromosomal subAB2 operon within an E. coli ST58 strain IBS28 (ONT:H25) taken from a wild ibis nest at an inland wetland in New South Wales, Australia. Here we show the subAB2 toxin operon comprised part of a 140 kb tRNA–Phe chromosomal island that co-hosted tia, encoding an outer-membrane protein that confers an adherence and invasion phenotype and additional virulence and accessory genetic content that potentially originated from known virulence island SE-PAI. This island shared a common evolutionary history with a secondary 90 kb tRNA–Phe pathogenicity island that was presumably generated via a duplication event. IBS28 is closely related [200 single-nucleotide polymorphisms (SNPs)] to four North American ST58 strains. The close relationship between North American isolates of ST58 and IBS28 was further supported by the identification of the only copy of a unique variant of IS26 within the O-antigen gene cluster. Strain ISB28 may be a historically important E. coli ST58 genome sequence hosting a progenitor pathogenicity island encoding subAB.

Thermodynamic analysis of the complexation of quinacrine with tRNAPhe

Abstract The thermodynamics of the interaction of the acridine derivative quinacrine with tRNA Phe was studied using isothermal titration calorimetry. The binding was monophasic and exothermic. At (298.15 ± 0.01) K the equilibrium constant was calculated to be (1.76 ± 0.70)·10 5 M −1 . The binding stoichiometry was (0.15 ± 0.04). The binding was predominantly enthalpy driven (Δ H o = −28.39 ± 0.60 kJ·mol −1 ). Salt dependent ITC studies followed by dissection of the standard molar Gibbs energy change showed that the binding reaction was driven by both polyelectrolytic and non-polyelectrolytic forces with the non-polyelectrolytic forces being dominant. Temperature dependent ITC studies showed that the binding became increasingly enthalpy driven with rise in temperature. The binding was characterized by negative standard molar heat capacity change and enthalpy-entropy compensation behaviour.

Effects of polyamines from Thermus thermophilus, an extreme-thermophilic eubacterium, on tRNA methylation by tRNA (Gm18) methyltransferase (TrmH).

Thermus thermophilus is an extreme-thermophilic eubacterium, which grows at a wide range of temperatures (50-83°C). This thermophile produces various polyamines including long and branched polyamines. In tRNAs from T. thermophilus, three distinct modifications, 2'-O-methylguanosine at position 18 (Gm18), 5-methyl-2-thiouridine at position 54 and N(1)-methyladenosine at position 58, are assembled at the elbow region to stabilize the L-shaped tRNA structure. However, the structures of unmodified tRNA precursors are disrupted at high temperatures. We hypothesize that polyamine(s) might have a positive effect on the modification process of unmodified tRNA transcript. We investigated the effects of eight polyamines on Gm18 formation in the yeast tRNA(Phe) transcript by tRNA (Gm18) methyltransferase (TrmH). Higher concentrations of linear polyamines inhibited TrmH activity at 55°C, while optimum concentration increased TrmH activity at 45-75°C. Exceptionally, caldohexamine, a long polyamine, did not show any positive effect on the TrmH activity at 55°C. However, temperature-dependent experiments revealed that 1 mM caldohexamine increased TrmH activity at 60-80°C. Furthermore, 0.25 mM tetrakis(3-aminopropy)ammonium, a branched polyamine, increased TrmH activity at a broad range of temperatures (40-85°C). Thus, caldohexamine and tetrakis(3-aminopropy)ammonium were found to enhance the TrmH activity at high temperatures.

Combined mitochondrial DNA analysis of the Mesopotamian spiny eel, Mastacembelus mastacembelus (Banks & Solander 1794), and its phylogenetic position

Nucleotide sequences of the 12S rRNA, 16S rRNA, tRNA Phe and tRNA Val genes of mtDNA of the Mesopotamian spiny eel, M. mastacembelus was determined for the first time. The comparison of the three populations of Mesopotamian spiny eel from Turkish part of the Tigris basin based on the obtained combined mitochondrial DNA was performed. Based on the results, no differences were determined and the identity found to be 100% among three populations. Furthermore, the obtained results from molecular methods were compared with morphological findings to validate the position of the studied populations of M. mastacembelus . In addition, the phylogenetic position of the Mesopotamian spiny eel was examined among the Mastacembelidae and Synbranchioformes based on 12S rRNA and 16S rRNA. The constructed phylogenetic relationship between M. mastacembelus and some other members of Synbranchioformes order supported their taxonomic hierarchy.

Complete mitochondrial genome of the chocolate hind Cephalopholis boenak (Pisces: Perciformes)

The complete mitochondrial (mt) genome of the chocolate hind Cephalopholis boenak was obtained in this study, which is the first complete mitogenome in the genus Cephalopholis. The circular mtDNA molecule was 16 771 bp in size and the overall nucleotide composition of the H-strand was 29.58% A, 27.36% T, 15.84% G, and 27.22% C, with an A + T bias. The complete mitogenome encoded 13 protein-coding genes, 2 rRNAs, 22 tRNAs and 1 control region (D-loop), with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. The control region located between the tRNA Pro and tRNA Phe genes and the length was 1 064 bp, rich in A + T (70.11%); it was composed of 13 complete continuity tandem repeat units, 5′-TACATATCTATGTACTTAA-3′, and one imperfect tandem repeat.

ONIOM and ab-initio calculations on the mechanism of uncatalyzed peptide bond formation

Finding a proper transition structure for the peptide bond formation process can lead one to a better understanding of the role of ribosome in catalyzing this reaction. Using computer simulations, we performed the potential energy surface scan on the ester bond dissociation of P-site aminoacyl-tRNA and the peptide bond formation of P-site and A-site amino acids. The full fragments of initiator tRNA(i)(met) and elongator tRNA(phe) are attached to both cognate and non-cognate amino acids as the P-site substrate. The A-site amino acid for all four calculations is methionine. We used ONIOM calculations to reduce the computational cost. Our study illustrates the reduced rate of peptide bond formation for misacylated tRNA(i)(met) in the absence of ribosomal bases. The misacylated elongator tRNA(phe), however, did not show any difference in its PES compared with that for the phe-tRNA(phe). This demonstrates the structural specification of initiator tRNA(i)(met) for the amino acids side chain.

The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning

RNase P is an RNA-based enzyme primarily responsible for 5′-end pre-tRNA processing. A structure of the bacterial RNase P holoenzyme in complex with tRNAPhe revealed the structural basis for substrate recognition, identified the active site location, and showed how the protein component increases functionality. The active site includes at least two metal ions, a universal uridine (U52), and P RNA backbone moieties, but it is unclear whether an adjacent, bacterially conserved protein loop (residues 52–57) participates in catalysis. Here, mutagenesis combined with single-turnover reaction kinetics demonstrate that point mutations in this loop have either no or modest effects on catalytic efficiency. Similarly, amino acid changes in the ‘RNR’ region, which represent the most conserved region of bacterial RNase P proteins, exhibit negligible changes in catalytic efficiency. However, U52 and two bacterially conserved protein residues (F17 and R89) are essential for efficient Thermotoga maritima RNase P activity. The U52 nucleotide binds a metal ion at the active site, whereas F17 and R89 are positioned >20 Å from the cleavage site, probably making contacts with N−4 and N−5 nucleotides of the pre-tRNA 5′-leader. This suggests a synergistic coupling between transition state formation and substrate positioning via interactions with the leader.

Crystal structures of the tRNA:m 2 G6 methyltransferase Trm14/TrmN from two domains of life

Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNAPhe at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m2G6 (N2-methylguanosine) MTase TTCTrmN from Thermus thermophilus and its ortholog PfTrm14 from Pyrococcus furiosus. Structures of PfTrm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. TTCTrmN and PfTrm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNAPhe of T. thermophilus and via site-directed mutagenesis.

Partial tandem duplication of mtDNA–tRNA Phe impairs mtDNA translation in late-onset mitochondrial myopathy

An 80-year-old woman (PI) has been suffering of late onset progressive weakness and wasting of lower-limb muscles, accompanied by high creatine kinase levels in blood. A muscle biopsy, performed at 63 years, showed myopathic features with partial deficiency of cytochrome c oxidase. A second biopsy taken 7 years later confirmed the presence of a mitochondrial myopathy but also of vacuolar degeneration and other morphological features resembling inclusion body myopathy. Her 46-year-old daughter (PII) and 50-year-old son (PIII) are clinically normal, but the creatine kinase levels were moderately elevated and the EMG was consistently myopathic in both. Analysis of mitochondrial DNA sequence revealed in all three patients a novel, homoplasmic 15 bp tandem duplication adjacent to the 5′ end of mitochondrial tRNA Phe gene, encompassing the first 11 nucleotides of this gene and the four terminal nucleotides of the adjacent D-loop region. Both mutant fibroblasts and cybrids showed low oxygen consumption rate, reduced mitochondrial protein synthesis, and decreased mitochondrial tRNA Phe amount. These findings are consistent with an unconventional pathogenic mechanism causing the tandem duplication to interfere with the maturation of the mitochondrial tRNA Phe transcript.

L-DOPA Ropes in tRNAPhe

L-DOPA is the most commonly prescribed drug for the treatment of Parkinson's disease. Here, Moor etal. (2011) report that phenylalanyl-tRNA synthetase catalyzes the misacylation of tRNA(Phe) by L-DOPA, suggesting that it may contribute to the elevated levels of L-DOPA-containing proteins found in patients treated with this drug.

Analysis of the complete mitochondrial genome sequence of Pielomastax zhengi

The complete mitochondrial genome sequence of Pielomastax zhengi was determined by using long PCR and conserved primer walking approaches. The results showed that the entire mitochondrial genome of Pielomastax zhengi is 15 602 bp long with A+T content of 71.8%. All 37 genes are conserved in the positions observed in those of Locusta migratoria. All the genes are closely assembled by leaving 10 intergenic spacers in between. Those intergenic spacers are 47 bp in total (excluding the A+T rich region), with individual size ranges from 1 bp to 20 bp. In addition, there are a total of 52 bp overlapping sequences among 14 genes, ranging from 1-8 bp. All protein-coding genes start with a typical initiation codon in insects, ATN. Twelve protein-coding genes use the usual TAA and TAG termination codons, whereas, the ND5 genes have an incomplete termination codon (T). Except the tRNASer (AGN), whose DHU arm is absent; all the other 21 tRNA genes have typical clover-leaf secondary structures. But in Pielomastax zhengi, the secondary structures of five tRNA (tRNA(Cys), tRNA(Lys), tRNA(Phe), tRNA(Pro), tRNA(Arg)) genes can not be predicted by the conventional methods as they have only 3-4, rather than 5 base pairs in the TψC arm, while, the tRNA(Lys) and tRNA(Arg) have only 4 base pairs in the anticode arm. The predicted secondary structures of lrRNA and srRNA have 6 domains with 44 helices and 3 domains with 30 helices, respectively. The results of the rRNA secondary structure comparison showed that Pielomastax zhengi is more closely related with Locusta migratoria tibetensis than Ruspolia dubia. Like most insects, the mitochondrial genome of Pielomastax zhengi has a non-coding A+T rich region containing a polythymidine stretch, which may be involved in the replication and/or translation initiation of other genes.

The complete mitochondrial genome of Macrobrachium nipponense

The complete mitochondrial (mt) genome sequence plays an important role in the accurate determination of phylogenetic relationships among metazoans. Herein, we determined the complete mt genome sequence, structure and organization of Macrobrachium nipponense ( M. nipponense) (GenBank ID: NC_015073.1) and compared it to that of Macrobrachium lanchesteri ( M. lanchesteri) and Macrobrachium rosenbergii ( M. rosenbergii). The 15,806 base pair (bp) M. nipponense mt genome, which is comprised of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs), is slightly larger than that of M. lanchesteri (15,694 bp, GenBank ID: NC_012217.1) and M. rosenbergii (15,772 bp, GenBank ID: NC_006880.1). The M. nipponense genome contains a high AT content (66.0%), which is a common feature among metazoan mt genomes. Compared with M. lanchesteri and M. rosenbergii, we found a peculiar non-coding region of 950 bp with a microsatellite-like (TA) 6 element and many hairpin structures. The 13 PCGs are comprised of a total of 3707 codons, excluding incomplete termination codons, and the most frequently used amino acid is Leu (16.0%). The predicted start codons in the M. nipponense mt genome include ATG, ATC and ATA. Seven PCGs use TAA as a stop codon, whereas two use TAG, three use T and only one uses TA. Twenty-three of the genes are encoded on the L strand, and ND1, ND4, ND5, ND4L, 12S rRNA, 16S rRNA, tRNA His , tRNA Pro , tRNA Phe , tRNA Val , tRNA Gln , tRNA Cys , tRNA Tyr and a tRNA Leu are encoded on the H strand. The two rRNAs of M. nipponense and M. rosenbergii are encoded on the H strand, whereas the M. lanchesteri rRNAs are encoded on the L stand.

Prediction of folding nuclei in tRNA molecules

Prediction of folding nuclei in RNA molecules allows one to look in a new way at the problem of possible RNA base sequence folding and at problems associated with incorrect RNA folding, as well as at RNA structure stability. We have chosen a model and energy parameters for description of RNA structure. The algorithm for studying processes including protein folding/unfolding was successfully applied to calculations on tRNA. Four tRNA molecules were considered whose structures were obtained in the free state (tRNA(Phe), tRNA(Asp), tRNA(fMet), and tRNA(Lys)). The calculated Φ-values for tRNA molecules correlate with experimental data showing that nucleotide residues in the D and T hairpin regions are involved in tRNA structure last, or more exactly, they are not included in the tRNA folding nucleus. High Φ-values in the anticodon hairpin region show that the nucleus of tRNA folding is localized just in that place.

The Role of L1 Stalk–tRNA Interaction in the Ribosome Elongation Cycle

The ribosomal L1 stalk is a mobile structure implicated in directing tRNA movement during translocation through the ribosome. This article investigates three aspects of L1 stalk–tRNA interaction. First, by combining data from cryo electron microscopy, X-ray crystallography, and molecular dynamics simulations through the molecular dynamics flexible fitting method, we obtained atomic models of different tRNAs occupying the hybrid P/E state interacting with the L1 stalk. These models confirm the assignment of fluorescence resonance energy transfer states from previous single-molecule investigations of L1 stalk dynamics. Second, the models reconcile how initiator tRNA fMet interacts less strongly with the L1 stalk compared to elongator tRNA Phe, as seen in previous single-molecule experiments. Third, results from a simulation of the entire ribosome in which the L1 stalk is moved from a half-closed conformation to its open conformation are found to support the hypothesis that L1 stalk opening is involved in tRNA release from the ribosome.

ICEEc2, a New Integrative and Conjugative Element Belonging to the pKLC102/PAGI-2 Family, Identified inEscherichia coliStrain BEN374

The diversity of the Escherichia coli species is in part due to the large number of mobile genetic elements that are exchanged between strains. We report here the identification of a new integrative and conjugative element (ICE) of the pKLC102/PAGI-2 family located downstream of the tRNA gene pheU in the E. coli strain BEN374. Indeed, this new region, which we called ICEEc2, can be transferred by conjugation from strain BEN374 to the E. coli strain C600. We were also able to transfer this region into a Salmonella enterica serovar Typhimurium strain and into a Yersinia pseudotuberculosis strain. This transfer was then followed by the integration of ICEEc2 into the host chromosome downstream of a phe tRNA gene. Our data indicated that this transfer involved a set of three genes encoding DNA mobility enzymes and a type IV pilus encoded by genes present on ICEEc2. Given the wide distribution of members of this family, these mobile genetic elements are likely to play an important role in the diversification of bacteria.

ChemInform Abstract: Studies Towards the Synthesis of the Hypermodified Nucleoside of Rat Liver Phenylalanine Transfer Ribonucleic Acid: Improved Synthesis of the Base β-Hydroxywybutine.

An improved synthesis of the key intermediates (3 and 8) for the synthesis of β-hydroxywybutines [[R-(R * ,S * )]- and [S-(R * ,R * )]-4], the most probable structures for the minor base from rat liver tRNA Phe , has been achieved by the Wittig reaction between 1-benzyl-7-formylwye (1) and the phosphorane derived from (R)-2-[(methoxycarbonyl)amino]-3-(triphenylphosphonio)propanoate (10), followed by methylation, OsO 4 oxidation, and cyclocondensation with COCl 2 in the presence of pyridine. The racemic forms of β-hydroxywybutines [(R * ,S * )- and (R * ,R * )-4], which were required for the determination of the optical purity of [R-(R * ,S * )]- and [S-(R * ,R * )]-4 by means of chiral HPLC, were conveniently prepared through pyrolysis of the cyclic carbonate 3 followed by NaBH 4 reduction and catalytic hydrogenolysis. The samples of [R-(R * ,S * )]- and [S-(R * ,R * )]-4 were thus shown to be optically pure.

The complete mitochondrial genome of the mantid shrimp Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda): Novel non-coding regions features and phylogenetic implications of the Stomatopoda

The complete mitochondrial (mt) genome sequence of Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda) was determined; a circular molecule of 15,783 bp in length. The gene content and arrangement are consistent with the pancrustacean ground pattern. The mt control region of O. oratoria is characterized by no GA-block near the 3′ end and different position of [TA(A)]n-blocks compared with other reported Stomatopoda species. The sequence of the second hairpin structure is relative conserved which suggests this region may be a synapomorphic character for the Stomatopoda. In addition, a relative large intergenic spacer (101 bp) with higher A + T content than that in control region was identified between the tRNA Glu and tRNA Phe genes. Phylogenetic analyses based on the current dataset of complete mt genomes strongly support the Stomatopoda is closely related to Euphausiacea. They in turn cluster with Penaeoidea and Caridea clades while other decapods form a separate group, which rejects the monophyly of Decapoda. This challenges the suitability of Stomatopoda as an outgroup of Decapoda in phylogenetic analyses. The basal position of Stomatopoda within Eumalacostraca according to the morphological characters is also questioned.

Of Least Concern? Systematics of a cryptic species complex: Limnonectes kuhlii (Amphibia: Anura: Dicroglossidae)

Several recent studies have demonstrated that a tremendous amount of biological diversity can be masked by phenotypic similarity in a cryptic species complex. It has been speculated that the widely distributed and relatively common Southeast Asian frog Limnonectes kuhlii represents a complex of multiple species. The phylogeny within the L. kuhlii Complex is estimated in this study based on approximately 2400 bp of mtDNA data (tRNA Phe, 12S, tRNA Val, and 16S genes) from 244 individuals representing multiple populations from throughout the known distribution of this anuran. Analyses are conducted using parsimony, maximum likelihood and Bayesian methods. The results suggest that what has been recognized historically as a single species is a complex of more than 22 distinct evolutionary lineages, 16 of which are currently subsumed under the nominal L. kuhlii. Several cases of sympatric lineages were detected, and in all cases co-occurring lineages were not each other’s closest relatives.