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Sample records for 5prime trna halves

  1. The heteromeric Nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge–helix–bulge motifs of joined tRNA halves

    PubMed Central

    Randau, Lennart; Calvin, Kate; Hall, Michelle; Yuan, Jing; Podar, Mircea; Li, Hong; Söll, Dieter

    2005-01-01

    Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5′ and 3′ tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge–helix–bulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate. PMID:16330750

  2. Angiogenin-cleaved tRNA halves interact with cytochrome c, protecting cells from apoptosis during osmotic stress.

    PubMed

    Saikia, Mridusmita; Jobava, Raul; Parisien, Marc; Putnam, Andrea; Krokowski, Dawid; Gao, Xing-Huang; Guan, Bo-Jhih; Yuan, Yiyuan; Jankowsky, Eckhard; Feng, Zhaoyang; Hu, Guo-fu; Pusztai-Carey, Marianne; Gorla, Madhavi; Sepuri, Naresh Babu V; Pan, Tao; Hatzoglou, Maria

    2014-07-01

    Adaptation to changes in extracellular tonicity is essential for cell survival. However, severe or chronic hyperosmotic stress induces apoptosis, which involves cytochrome c (Cyt c) release from mitochondria and subsequent apoptosome formation. Here, we show that angiogenin-induced accumulation of tRNA halves (or tiRNAs) is accompanied by increased survival in hyperosmotically stressed mouse embryonic fibroblasts. Treatment of cells with angiogenin inhibits stress-induced formation of the apoptosome and increases the interaction of small RNAs with released Cyt c in a ribonucleoprotein (Cyt c-RNP) complex. Next-generation sequencing of RNA isolated from the Cyt c-RNP complex reveals that 20 tiRNAs are highly enriched in the Cyt c-RNP complex. Preferred components of this complex are 5' and 3' tiRNAs of specific isodecoders within a family of isoacceptors. We also demonstrate that Cyt c binds tiRNAs in vitro, and the pool of Cyt c-interacting RNAs binds tighter than individual tiRNAs. Finally, we show that angiogenin treatment of primary cortical neurons exposed to hyperosmotic stress also decreases apoptosis. Our findings reveal a connection between angiogenin-generated tiRNAs and cell survival in response to hyperosmotic stress and suggest a novel cellular complex involving Cyt c and tiRNAs that inhibits apoptosome formation and activity. PMID:24752898

  3. Several RNase T2 enzymes function in induced tRNA and rRNA turnover in the ciliate Tetrahymena.

    PubMed

    Andersen, Kasper L; Collins, Kathleen

    2012-01-01

    RNase T2 enzymes are produced by a wide range of organisms and have been implicated to function in diverse cellular processes, including stress-induced anticodon loop cleavage of mature tRNAs to generate tRNA halves. Here we describe a family of eight RNase T2 genes (RNT2A-RNT2H) in the ciliate Tetrahymena thermophila. We constructed strains lacking individual or combinations of these RNT2 genes that were viable but had distinct cellular and molecular phenotypes. In strains lacking only one Rnt2 protein or lacking a subfamily of three catalytically inactive Rnt2 proteins, starvation-induced tRNA fragments continued to accumulate, with only a minor change in fragment profile in one strain. We therefore generated strains lacking pairwise combinations of the top three candidates for Rnt2 tRNases. Each of these strains showed a distinct starvation-specific profile of tRNA and rRNA fragment accumulation. These results, the delineation of a broadened range of conditions that induce the accumulation of tRNA halves, and the demonstration of a predominantly ribonucleoprotein-free state of tRNA halves in cell extract suggest that ciliate tRNA halves are degradation intermediates in an autophagy pathway induced by growth arrest that functions to recycle idle protein synthesis machinery.

  4. Multiple Layers of Stress-Induced Regulation in tRNA Biology

    PubMed Central

    Huang, Hsiao-Yun; Hopper, Anita K.

    2016-01-01

    tRNAs are the fundamental components of the translation machinery as they deliver amino acids to the ribosomes during protein synthesis. Beyond their essential function in translation, tRNAs also function in regulating gene expression, modulating apoptosis and several other biological processes. There are multiple layers of regulatory mechanisms in each step of tRNA biogenesis. For example, tRNA 3′ trailer processing is altered upon nutrient stress; tRNA modification is reprogrammed under various stresses; nuclear accumulation of tRNAs occurs upon nutrient deprivation; tRNA halves accumulate upon oxidative stress. Here we address how environmental stresses can affect nearly every step of tRNA biology and we describe the possible regulatory mechanisms that influence the function or expression of tRNAs under stress conditions. PMID:27023616

  5. Oligomerizations of deoxyadenosine bis-phosphates and of their 3-prime-5-prime, 3-prime-3-prime, and 5-prime-5-prime dimers - Effects of a pyrophosphate-linked, poly(T) analog

    NASA Technical Reports Server (NTRS)

    Visscher, J.; Bakker, C. G.; Schwartz, Alan W.

    1990-01-01

    The effect of a 3-prime-5-prime pyrophosphate-linked oligomer of pTp on oligomerizations of pdAp and of its 3-prime-5-prime, 3-prime-3-prime, and 5-prime-5-prime dimers was investigated, using HPLC to separate the reaction mixtures; peak detection was by absorbance monitoring at 254 nm. It was expected that the dimers would form stable complexes with the template, with the degree of stability depending upon the internal linkage of each dimer. It was found that, although the isomers differ substantially in their oligomerization behavior in the absence of template, the analog-template catalyzes the oligomerization to about the same extent in all three cases.

  6. 7 CFR 51.1437 - Size classifications for halves.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... halves per pound shall be based upon the weight of half-kernels after all pieces, particles and dust... of numbers of halves per pound. For example, “400” or “600-700”. (c) Tolerance for count per pound...: (1) When an exact number of halves per pound is specified, the actual count per pound may vary...

  7. 7 CFR 51.1437 - Size classifications for halves.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... halves per pound shall be based upon the weight of half-kernels after all pieces, particles and dust... of numbers of halves per pound. For example, “400” or “600-700”. (c) Tolerance for count per pound...: (1) When an exact number of halves per pound is specified, the actual count per pound may vary...

  8. Shaping tRNA

    ERIC Educational Resources Information Center

    Priano, Christine

    2013-01-01

    This model-building activity provides a quick, visual, hands-on tool that allows students to examine more carefully the cloverleaf structure of a typical tRNA molecule. When used as a supplement to lessons that involve gene expression, this exercise reinforces several concepts in molecular genetics, including nucleotide base-pairing rules, the…

  9. 7 CFR 51.1433 - U.S. Commercial Halves.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false U.S. Commercial Halves. 51.1433 Section 51.1433... STANDARDS) United States Standards for Grades of Shelled Pecans Grades § 51.1433 U.S. Commercial Halves. The requirements for this grade are the same as those for U.S. No. 1 Halves except: (a) For quality: (1)...

  10. 5[prime] to 3[prime] nucleic acid synthesis using 3[prime]-photoremovable protecting group

    DOEpatents

    Pirrung, M.C.; Shuey, S.W.; Bradley, J.C.

    1999-06-01

    The present invention relates, in general, to a method of synthesizing a nucleic acid, and, in particular, to a method of effecting 5[prime] to 3[prime] nucleic acid synthesis. The method can be used to prepare arrays of oligomers bound to a support via their 5[prime] end. The invention also relates to a method of effecting mutation analysis using such arrays. The invention further relates to compounds and compositions suitable for use in such methods.

  11. Effect of cold exposure on thyroxine 5 prime -deiodinase activity in iron-deficient rats

    SciTech Connect

    Brigham, D.E.; Beard, J.L. )

    1991-03-11

    When exposed to cold, severely anemic iron-deficient (ID) rats become hypothermic, fail to adequately increase metabolic rate, and have lower interscapular brown adipose tissue (IBAT) thyroxine 5{prime}-deiodinase (5{prime}-DI) activity compared to control (CN) rats. Less severely anemic rats and CN rats were exposed to 4C for 6h. Rectal temperatures were measured hourly, and VO{sub 2} was measured both prior to and throughout the cold exposure period. IBAT 5{prime}-DI activity was measured in cold-exposed ID and CN rats and compared to ID and CN rats that were not cold exposed. During cold exposure, both ID and CN rats increased metabolic rate similarly. IBAT 5{prime}-DI activity also increased similarly in both groups after cold exposure. These results show that moderately anemic DI rats acutely increase metabolic rate and IBAT 5{prime}-DI activity in the cold. This suggests brown fat production of thyroid hormone is not limiting thermoregulatory performance.

  12. Effect of dietary selenium on the 5 prime -deiodinase activity of washed microsomes from rat liver

    SciTech Connect

    Vadhanavikit, S.; Ganther, H.E. )

    1991-03-11

    Male Sprague Dawley rats were fed a Torula yeast-based diet containing 0.1 ppm Se as selenite (+Se) or the diet without selenium supplementation ({minus}Se). At 6 months, rats were sacrificed and liver microsomes and cytosol were prepared. Glutathione peroxidase activity of (+SE) and ({minus}Se) cytosol was 0.86 {plus minus} 0.07 and 0.02 {plus minus} 0.01 U/mg, respectively. Microsomal 5{prime}-deiodinase, Type I (5{prime}-DI) was assayed in the presence of 3 mM DTT using ({sup 125}I) reverse triiodothyronine (rT{sub 3}) as substrate and the released {sup 125}I{sup {minus}} was determined. 5{prime}-DI activity of (+Se) and ({minus}Se) microsomes was 3.99 {plus minus} 0.29 and 0.43 {plus minus} 0.12 U/mg, respectively. Mixing experiments showed that (a) cytosol from (+Se) animals had very little or no stimulating effect on 5{prime}-DI activity of ({minus}Se) microsomes, and (b) ({minus}Se) cytosol as well as ({minus}Se) microsomes had no inhibitory effect on (+Se) microsomes. It is concluded that 5{prime}-DI activity is greatly decreased in microsomes of Se-deficient rats, and is not restored to normal by the addition of cytosol from Se-adequate animals. These results confirm and extend the authors' previous report of reduced 5{prime}-DI activity in Se-deficient rats when measured as the conversion of thyroxine to T{sub 3} in whole homogenates.

  13. 2nd Antibiotic Halves C-Section Infection Rate

    MedlinePlus

    ... gov/news/fullstory_161230.html 2nd Antibiotic Halves C-Section Infection Rate: Study Two medications are better ... delivery, Andrews said. Overall, about 12 percent of C-sections result in an infection, according to background ...

  14. HUNTER 20 MATCHPLATE MOLDING MACHINE 'SQUEEZING' BOTH HALVES OF A ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    HUNTER 20 MATCHPLATE MOLDING MACHINE 'SQUEEZING' BOTH HALVES OF A MOLD SURROUNDING A MATCHPLATE PATTERN, DENNIS GRAY OPERATOR. - Southern Ductile Casting Company, Casting, 2217 Carolina Avenue, Bessemer, Jefferson County, AL

  15. A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair.

    PubMed

    Meineke, Birthe; Kast, Alene; Schwer, Beate; Meinhardt, Friedhelm; Shuman, Stewart; Klassen, Roland

    2012-09-01

    PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases. PMID:22836353

  16. Modulation of 2{prime}-5{prime} oligoadenylate synthetase by environmental stress in the marine sponge Geodia cydonium

    SciTech Connect

    Schroeder, H.C.; Wiens, M.; Mueller, W.E.G.; Kuusksalu, A.; Kelve, M.

    1997-07-01

    Recently the authors established the presence of relatively high amounts of 2{prime}-5{prime} oligoadenylates (2{prime}-5{prime} A) and 2{prime}-5{prime} oligoadenylate synthetase (2{prime}-5{prime} A synthetase) in the marine sponge Geodia cydonium. Here they determined by applying radioimmunoassay and high-performance liquid chromatographical methods that the concentration of 2{prime}-5{prime} A synthetase change following exposure of G. cydonium tissue to environmental stress. The 2{prime}-5{prime} A content and the activity of 2{prime}-5{prime} A synthetase, present in crude sponge extract, increase by up to three-fold after treating sponge cubes for 2 h with natural stressors including heat shock (26 C), cold shock (6 C), pH shock (pH 6), and hypertonic shock and subsequent incubation for 18 h under ambient conditions (16 C). No response was observed after exposure of sponges to an alkaline (pH 10) or hypotonic environment. Similar changes have been found for the expression of heat shock protein HSP70 in G. cydonium. These results show that 2{prime}-5{prime} A in sponges may be useful as a novel biomarker for environmental monitoring.

  17. Circularly permuted tRNA genes: their expression and implications for their physiological relevance and development

    PubMed Central

    Soma, Akiko

    2014-01-01

    A number of genome analyses and searches using programs that focus on the RNA-specific bulge-helix-bulge (BHB) motif have uncovered a wide variety of disrupted tRNA genes. The results of these analyses have shown that genetic information encoding functional RNAs is described in the genome cryptically and is retrieved using various strategies. One such strategy is represented by circularly permuted tRNA genes, in which the sequences encoding the 5′-half and 3′-half of the specific tRNA are separated and inverted on the genome. Biochemical analyses have defined a processing pathway in which the termini of tRNA precursors (pre-tRNAs) are ligated to form a characteristic circular RNA intermediate, which is then cleaved at the acceptor-stem to generate the typical cloverleaf structure with functional termini. The sequences adjacent to the processing site located between the 3′-half and the 5′-half of pre-tRNAs potentially form a BHB motif, which is the dominant recognition site for the tRNA-intron splicing endonuclease, suggesting that circularization of pre-tRNAs depends on the splicing machinery. Some permuted tRNAs contain a BHB-mediated intron in their 5′- or 3′-half, meaning that removal of an intron, as well as swapping of the 5′- and 3′-halves, are required during maturation of their pre-tRNAs. To date, 34 permuted tRNA genes have been identified from six species of unicellular algae and one archaeon. Although their physiological significance and mechanism of development remain unclear, the splicing system of BHB motifs seems to have played a key role in the formation of permuted tRNA genes. In this review, current knowledge of circularly permuted tRNA genes is presented and some unanswered questions regarding these species are discussed. PMID:24744771

  18. Characterization of Flavonoid 3[prime],5[prime]-Hydroxylase in Microsomal Membrane Fraction of Petunia hybrida Flowers.

    PubMed Central

    Menting, JGT.; Scopes, R. K.; Stevenson, T. W.

    1994-01-01

    We have detected a flavonoid 3[prime],5[prime]-hydroxylase (F3[prime],5[prime]H) in the microsomal fraction of Petunia hybrida flowers. Activity varied with the development of flowers, peaking immediately prior to and during anthesis, but was absent in mature flowers. F3[prime],5[prime]H activity in flower extracts from genetically defined floral color mutants correlated strictly with the genotypes Hf1 and Hf2. No activity was detected in flowers from mutants homozygous recessive for both alleles. F3[prime],5[prime]H activity was dependent on NADPH and molecular oxygen; there was only slight activity with NADH. The enzyme catalyzes the hydroxylation of 5,7,4[prime]-trihydroxyflavonone at the 3[prime] and 5[prime] positions, and of 5,7,3[prime],4[prime]-tetrahydroxyflavonone and dihydroquercetin at the 5[prime] position. Hydroxylase activity was inhibited by plant growth regulators (1-aminobenzotriazole and tetcyclacis) and by CO, N-ethylmaleimide, diethyldithiocarbamate, and cytochrome (Cyt) c. Activity was not affected by diethylpyrocarbonate or phenylmethylsulfonyl fluoride, but was enhanced by 2-mercaptoethanol. A polyclonal antibody that inhibits higher plant NADPH-Cyt P450 reductase inhibited the F3[prime],5[prime]H. The data are consistent with the suggestion that the P. hybrida F3[prime],5[prime]H is a monooxygenase consisting of a Cyt P450 and a NADPH-Cyt P-450 reductase. Cyts P450 were detected in microsomal membranes and in solubilized detergent extracts of these membranes. F3[prime],5[prime]H activity was sensitive to low concentrations of all detergents tested, and therefore solubilization of the active enzyme was not achieved. Reaction products other than flavanones were observed in F3[prime],5[prime]H assays and these may be formed by enzymic oxidation of flavanones. The possibility of a microsomal flavone synthase of a type that has not been described in P. hybrida is discussed. PMID:12232356

  19. Delving Deeper: One Cut, Two Halves, Three Questions

    ERIC Educational Resources Information Center

    Ren, Guanshen

    2009-01-01

    A square can be divided into two equal parts with any cut through the center. The first question that arises is, Would any cut through the center of a regular polygon divide it into two equal parts? If not, the second question is, What kind of lines through the center of the polygon would cut it into two halves? However, many objects are not…

  20. Jobs, Skills and Incomes in Ghana: How Was Poverty Halved?

    ERIC Educational Resources Information Center

    Nsowah-Nuamah, Nicholas; Teal, Francis; Awoonor-Williams, Moses

    2012-01-01

    On the basis of official statistics, poverty has halved in Ghana over the period from 1991 to 2005. Our objective in this paper is to assess how far this fall was linked to the creation of better paying jobs and the increase in education. We find that earnings rose rapidly in the period from 1998 to 2005, by 64% for men and by 55% for women. While…

  1. Multichromosomal median and halving problems under different genomic distances

    PubMed Central

    Tannier, Eric; Zheng, Chunfang; Sankoff, David

    2009-01-01

    Background Genome median and genome halving are combinatorial optimization problems that aim at reconstructing ancestral genomes as well as the evolutionary events leading from the ancestor to extant species. Exploring complexity issues is a first step towards devising efficient algorithms. The complexity of the median problem for unichromosomal genomes (permutations) has been settled for both the breakpoint distance and the reversal distance. Although the multichromosomal case has often been assumed to be a simple generalization of the unichromosomal case, it is also a relaxation so that complexity in this context does not follow from existing results, and is open for all distances. Results We settle here the complexity of several genome median and halving problems, including a surprising polynomial result for the breakpoint median and guided halving problems in genomes with circular and linear chromosomes, showing that the multichromosomal problem is actually easier than the unichromosomal problem. Still other variants of these problems are NP-complete, including the DCJ double distance problem, previously mentioned as an open question. We list the remaining open problems. Conclusion This theoretical study clears up a wide swathe of the algorithmical study of genome rearrangements with multiple multichromosomal genomes. PMID:19386099

  2. 7 CFR 51.1430 - U.S. No. 1 Halves.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...) For size: (1) Halves are fairly uniform in size; (2) Halves conform to size classification or count specified; and, (3) Comply with tolerances for pieces, particles, and dust (see § 51.1437)....

  3. 7 CFR 51.1430 - U.S. No. 1 Halves.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...) For size: (1) Halves are fairly uniform in size; (2) Halves conform to size classification or count specified; and, (3) Comply with tolerances for pieces, particles, and dust (see § 51.1437)....

  4. Phorbol esters, protein kinase C, and thyroxine 5 prime -deiodinase in brown adipocytes

    SciTech Connect

    Barge, R.M.; Mills, I.; Silva, J.E.; Larsen, P.R. )

    1988-03-01

    Protein kinase C activity has been identified in the rat brown adipocyte. About 60% of this activity is found in the cytosolic fraction under basal conditions, and 12-O-tetradecanoylphorbol 13-acetate (TPA) causes a rapid shift from the cytosol to the particulate fraction. Norepinephrine and phenylephrine causes a similar redistribution that can be blocked by prazosin but not by alprenolol. {alpha}{sub 1}-Adrenergic agonists cause three- to fivefold stimulation of type 2 iodothyronine 5{prime}-deiodinase activity in brown adipocytes. TPA has no effect on basal deiodinase activity and reduces the response of the enzyme to {alpha}{sub 1}-adrenergic agonists. These results suggest that the translocation of protein kinase C from cytosol to particulate fraction is not sufficient to increase deiodinase activity but can modulate the {alpha}{sub 1}-adrenergic agonist-mediated responses in these cells.

  5. Eukaryotic tRNA paradox.

    PubMed

    Mitra, Sanga; Samadder, Arpa; Das, Pijush; Das, Smarajit; Chakrabarti, Jayprokas

    2015-01-01

    tRNAs are widely believed to segregate into two classes, I and II. Computational analysis of eukaryotic tRNA entries in Genomic tRNA Database, however, leads to new, albeit paradoxical, presence of more than a thousand class-I tRNAs with uncharacteristic long variable arms (V-arms), like in class-II. Out of 62,202 tRNAs from 69 eukaryotes, as many as 1431 class-I tRNAs have these novel extended V-arms, and we refer to them as paradoxical tRNAs (pxtRNAs). A great majority of these 1431 pxtRNA genes are located in intergenic regions, about 18% embedded in introns of genes or ESTs, and just one in 3'UTR. A check on the conservations of 2D and 3D base pairs for each position of these pxtRNAs reveals a few variations, but they seem to have almost all the known features (already known identity and conserved elements of tRNA). Analyses of the A-Box and B-Box of these pxtRNA genes in eukaryotes display salient deviations from the previously annotated conserved features of the standard promoters, whereas the transcription termination signals are just canonical and non-canonical runs of thymidine, similar to the ones in standard tRNA genes. There is just one such pxtRNA(ProAGG) gene in the entire human genome, and the availability of data allows epigenetic analysis of this human pxtRNA(ProAGG) in three different cell lines, H1 hESC, K562, and NHEK, to assess the level of its expression. Histone acetylation and methylation of this lone pxtRNA(ProAGG) gene in human differ from that of the nine standard human tRNA(ProAGG) genes. The V-arm nucleotide sequences and their secondary structures in pxtRNA differ from that of class-II tRNA. Considering these differences, hypotheses of alternative splicing, non-canonical intron and gene transfer are examined to partially improve the Cove scores of these pxtRNAs and to critically question their antecedence and novelty. PMID:25692737

  6. Methylated nucleosides in tRNA and tRNA methyltransferases

    PubMed Central

    Hori, Hiroyuki

    2014-01-01

    To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. PMID:24904644

  7. Effects of halving pesticide use on wheat production

    PubMed Central

    Hossard, L.; Philibert, A.; Bertrand, M.; Colnenne-David, C.; Debaeke, P.; Munier-Jolain, N.; Jeuffroy, M. H.; Richard, G.; Makowski, D.

    2014-01-01

    Pesticides pose serious threats to both human health and the environment. In Europe, farmers are encouraged to reduce their use, and in France a recent environmental policy fixed a target of halving the pesticide use by 2018. Organic and integrated cropping systems have been proposed as possible solutions for reducing pesticide use, but the effect of reducing pesticide use on crop yield remains unclear. Here we use a set of cropping system experiments to quantify the yield losses resulting from a reduction of pesticide use for winter wheat in France. Our estimated yield losses resulting from a 50% reduction in pesticide use ranged from 5 to 13% of the yield obtained with the current pesticide use. At the scale of the whole country, these losses would decrease the French wheat production by about 2 to 3 millions of tons, which represent about 15% of the French wheat export. PMID:24651597

  8. Novel adenosine 3 prime ,5 prime -cyclic monophosphate dependent protein kinases in a marine diatom

    SciTech Connect

    Lin, P.P.C.; Volcani, B.E. )

    1989-08-08

    Two novel adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg{sup 2+} and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser({sup 32}P)-Ser-Asn-Ala-Arg and have an apparent M{sub r} of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M{sub r} of about 78,000 is photolabeled with 8-azido({sup 32}P)cAMP and is also phosphorylated with ({gamma}-{sup 32}P)ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids.

  9. Halving the Casimir force with conductive oxides: Experimental details

    SciTech Connect

    Man, S. de; Heeck, K.; Iannuzzi, D.

    2010-12-15

    This work is an extended version of a paper published previously [S. de Man et al., Phys. Rev. Lett. 103, 040402 (2009)], where we presented measurements of the Casimir force between a gold-coated sphere and a plate coated with either gold or an indium tin oxide (I TO) layer. The experiment, which was performed in air, showed that I TO conducts sufficiently to prevent charge accumulation but is still transparent enough to halve the Casimir attraction when compared to gold. Here we report all the experimental details that, owing to the limited space available, were omitted in the previous article. We discuss the performance of our setup in terms of stability of the calibration procedure and reproducibility of the Casimir force measurement. We also introduce and demonstrate a technique to obtain the spring constant of our force sensor. Furthermore, we present a thorough description of the experimental method, a comprehensive explanation of data elaboration and error analysis, and a complete characterization of the dielectric function and of the surface roughness of the samples used in the actual experiment.

  10. RNA polymerase II pauses at the 5 prime end of the transcriptionally induced Drosophila hsp70 gene

    SciTech Connect

    O'Brien, T.; Lis, J.T. )

    1991-10-01

    An RNA polymerase II molecule is associated with the 5{prime} end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides. Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here the authors report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5{prime} end of hsp70 was transcribed approximately five times during the 25-min heat shock that they used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

  11. Stereochemical mechanisms of tRNA methyltransferases

    PubMed Central

    Hou, Ya-Ming; Perona, John J.

    2009-01-01

    Methylation of tRNA on the four canonical bases adds structural complexity to the molecule, and improves decoding specificity and efficiency. While many tRNA methylases are known, detailed insight into the catalytic mechanism is only available in a few cases. Of interest among all tRNA methylases is the structural basis for nucleotide selection, by which the specificity is limited to a single site, or broadened to multiple sites. General themes in catalysis include the basis for rate acceleration at highly diverse nucleophilic centers for methyl transfer, using S-adenosylmethionine as a cofactor. Studies of tRNA methylases have also yielded insights into molecular evolution, particularly in the case of enzymes that recognize distinct structures to perform identical reactions at the same target nucleotide. PMID:19944101

  12. Binding of nickel /II/ to 5-prime-nucleoside monophosphates and related compounds. [role in origin of life

    NASA Technical Reports Server (NTRS)

    Orenberg, J. B.; Kjos, K. M.; Winkler, R.; Link, J.; Lawless, J. G.

    1982-01-01

    The interactions of Ni(II) cation with a representative suite of purine bases and the respective nucleosides and nucleotides have been studied by ultraviolet difference spectroscopy. Apparent association constants were determined for each system at pH 7.0, using computer linear regression coupled with an iteration technique. The specificity of binding of Ni(2+) for the purine nucleotides studied at pH 7.0 was 5-prime-GMP greater than 5-prime-AMP; a similar ordering was also found for the respective nucleosides and bases. In this study binding was not observed for the suite of pyramidines used, although an Ni(2+) -cytidine complex has been observed (Fiskin and Beer, 1965). It was also found that Ni(2+) bound more strongly to the purine 5-prime-nucleotides than to the respective nucleosides and bases. These trends are explained in terms of metal-ligand bonds and available bonding positions on the ligands. A role for metal-ion-nucleotide types of complexes is suggested in the processes that might have given rise to the origin of life.

  13. Halving Student Loan Interest Rates Is Unaffordable and Ineffective. WebMemo No. 1308

    ERIC Educational Resources Information Center

    Riedl, Brian M.

    2007-01-01

    The House of Representatives will likely vote this week on a proposal to halve the 6.8 percent interest rate on subsidized student loans as part of the new congressional majority's 100-Hour agenda. This document presents six problems with halving student loan interest rates and argues that, rather than providing billions in new federal subsidies,…

  14. Comparison of the halving of tablets prepared with eccentric and rotary tablet presses.

    PubMed

    Sovány, T; Kása, P; Pintye-Hódi, K

    2009-01-01

    The aim of this study was to compare the densification of powder mixtures on eccentric and rotary tablet presses and to establish relationships with the halving properties of the resulting scored tablets. This is an important problem because the recent guidelines of EU require verification of the equal masses of tablet halves. The models of Walker, Heckel, and Kawakita were used to describe the powder densification on the two machines. The calculated parameters revealed that the shorter compression cycle of rotary machines results in poorer densification and lower tablet hardness at a given compression force. This is manifested in poorer halving properties, which are influenced mainly by the hardness. Better densification improves the halving even at lower tablet hardness. This demonstrates that these parameters can be good predictors of tablet halving properties. PMID:19381830

  15. Speeding Up Sigmatropic Shifts-To Halve or to Hold.

    PubMed

    Tantillo, Dean J

    2016-04-19

    Catalysis is common. Rational catalyst design, however, is at the frontier of chemical science. Although the histories of physical organic and synthetic organic chemistry boast key chapters involving [3s,3s] sigmatropic shifts, catalysis of these reactions is much less common than catalysis of ostensibly more complex processes. The comparative dearth of catalysts for sigmatropic shifts is perhaps a result of the perception that transition state structures for these reactions, like their reactants, are nonpolar and therefore not amenable to selective stabilization and its associated barrier lowering. However, as demonstrated in this Account, transition state structures for [3s,3s] sigmatropic shifts can in fact have charge distributions that differ significantly from those of reactants, even for hydrocarbon substrates, allowing for barriers to be decreased and rates increased. In some cases, differences in charge distribution result from the inclusion of heteroatoms at specific positions in reactants, but in other cases differences are actually induced by catalysts. Perhaps surprisingly, strategies for complexation of transition state structures that remain nonpolar are also possible. In general, the strategies for catalysis employed can be characterized as involving either mechanistic intervention, where a catalyst induces a change from the concerted mechanism expected for a [3s,3s] sigmatropic shift to a multistep process (cutting the transformation into halves or smaller pieces) whose overall barrier is decreased relative to the concerted process, or transition state complexation, where a catalyst simply binds (holds) more tightly to the transition state structure for a [3s,3s] sigmatropic shift than to the reactant, leading to a lower barrier in the presence of the catalyst. Both of these strategies can be considered to be biomimetic in that enzymes frequently induce multistep processes and utilize selective transition state stabilization for the steps involved

  16. Isolation of an insulin-like growth factor II cDNA with a unique 5 prime untranslated region from human placenta

    SciTech Connect

    Shen, Shujane; Daimon, Makoto; Wang, Chunyeh; Ilan, J. ); Jansen, M. )

    1988-03-01

    Human insulin-like growth factor II (IGF-II) cDNA from a placental library was isolated and sequenced. The 5{prime} untranslated region (5{prime}-UTR) sequence of this cDNA differs completely from that of adult human liver and has considerable base sequence identity to the same region of an IGF-II cDNA of a rat liver cell line, BRL-3A. Human placental poly(A){sup +} RNA was probed with either the 5{prime}-UTR of the isolated human placental IGF-II cDNA or the 5{prime}-UTR of the IGF-II cDNA obtained from adult human liver. No transcripts were detected by using the 5{prime}-UTR of the adult liver IGF-II as the probe. In contrast, three transcripts of 6.0, 3.2, and 2.2 kilobases were detected by using the 5{prime}-UTR of the placental IGF-II cDNA as the probe or the probe from the coding sequence. A fourth IGF-II transcript of 4.9 kilobases presumably containing a 5{prime}-UTR consisting of a base sequence dissimilar to that of either IGF-II 5{prime}-UTR was apparent. Therefore, IGF-II transcripts detected may be products of alternative splicing as their 5{prime}-UTR sequence is contained within the human IGF-II gene or they may be a consequence of alternative promoter utilization in placenta.

  17. 40 CFR 761.306 - Sampling 1 meter square surfaces by random selection of halves.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... shape of a circle as possible. For example, a square is closer to the shape of a circle than is a rectangle and a rectangle having a length to width ratio of 2:1 is closer to the shape of a circle than a... square shape the top/bottom halves have the same shape as the left/right halves when compared to a...

  18. 40 CFR 761.306 - Sampling 1 meter square surfaces by random selection of halves.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... shape of a circle as possible. For example, a square is closer to the shape of a circle than is a rectangle and a rectangle having a length to width ratio of 2:1 is closer to the shape of a circle than a... square shape the top/bottom halves have the same shape as the left/right halves when compared to a...

  19. 40 CFR 761.306 - Sampling 1 meter square surfaces by random selection of halves.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... shape of a circle as possible. For example, a square is closer to the shape of a circle than is a rectangle and a rectangle having a length to width ratio of 2:1 is closer to the shape of a circle than a... square shape the top/bottom halves have the same shape as the left/right halves when compared to a...

  20. Linkage disequilibrium in the insulin gene region: Size variation at the 5{prime} flanking polymorphism and bimodality among {open_quotes}Class I{close_quotes} alleles

    SciTech Connect

    McGinnis, R.E.; Spielman, R.S.

    1994-09-01

    The 5{prime} flanking polymorphism (5{prime}FP), a hypervariable region at the 5{prime} end of the insulin gene, has {open_quotes}class 1{close_quotes} alleles (650-900 bp long) that are in positive linkage disequilibrium with insulin-dependent diabetes mellitus (IDDM). The authors report that precise sizing of the 5{prime}FP yields a bimodal frequency distribution of class 1 allele lengths. Class 1 alleles belonging to the lower component (650-750 bp) of the bimodal distribution were somewhat more highly associated with IDDM than were alleles from the upper component (760-900 bp), but the difference was not statistically significant. They also examined 5{prime}FP length variation in relation to allelic variation at nearby polymorphisms. At biallelic RFLPs on both sides of the 5{prime}FP, they found that one allele exhibits near-total association with the upper component of the 5FP class 1 distribution. Such associations represent a little-known but potentially wide-spread form of linkage disequilibrium. In this type of disequilibrium, a flanking allele has near-complete association with a single mode of VNTR alleles whose lengths represent consecutive numbers of tandem repeats (CNTR). Such extreme disequilibrium between a CNTR mode and flanking alleles may originate and persist because length mutations at some VNTR loci usually add or delete only one or two repeat units. 22 refs., 5 figs., 6 tabs.

  1. Analysis of the 5{prime} junctions of R2 insertions with the 28S gene: Implications for non-LTR retrotransposition

    SciTech Connect

    George, J.A.; Burke, W.D.; Eickbush, T.H.

    1996-03-01

    R2 elements are non-long terminal repeat retrotransposable elements that insert into 28S rRNA genes of most insect species. The single open reading frame of R2 encodes a protein with both endonuclease activity, which cleaves the target site, and reverse transcriptase activity, which uses this cleavage to prime reverse transcription. This target-primed reverse transcription mechanism is also used by group II introns. Little is known of the mechanism by which the 5{prime} end of R2 is integrated after reverse transcription. We have determined the 5{prime} junction sequence of 94 R2 elements from 14 different species of Drosophila. Only 37% of the full-length elements contained precise 5{prime} junctions; the remainder contained deletions of the 28S gene and/or insertions of additional sequences. Because the 5{prime} junctions of truncated copies were similar to full-length elements, no sequences at the 5{prime} end of R2 appear to be required for element integration. A model in which the R2 reverse transcriptase is capable of switching templates from the R2 RNA transcript to the upstream 28S gene can best explain the observed 5{prime} junction sequences. This template jumping is analogous to the template switching of retroviral reverse transcriptases during formation of the double-stranded integration products. 44 refs., 5 figs.

  2. tRNA Biology in Mitochondria

    PubMed Central

    Salinas-Giegé, Thalia; Giegé, Richard; Giegé, Philippe

    2015-01-01

    Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation. PMID:25734984

  3. Binding of nuclear factors to the 5 prime -interferon consensus sequence of the HLA-A2 class I gene

    SciTech Connect

    Le Bouteiller, P.; Bogarad, L.D.; Roberts, M.R.; Ruddle, F.H. ); Barbosa, J.A. )

    1990-02-01

    To investigate the regulatory role of the conserved interferon consensus sequence (ICS) found in the 5{prime} flanking region of HLA class I genes, the authors studied the binding of nuclear proteins to the ICS of HLA-A2 gene (ICS-A2) by the gel shift assay. Nuclear extracts from several human cell lines expressing different levels of surface class I molecules reveal an ICS-A2-protein complex of similar mobility, the amount of which varies in a cell-type=dependent manner. In some cell lines, interferon-{gamma} treatment decreased the level of this complex. The overlapping enhancer A element also competes for the formation of this ICS-A2-protein complex. Footprinting and methylation interference analyses demonstrate that nuclear protein(s) protect specific sequences within the ICS-A2 element, suggesting that these protein(s) may represent interferon-sensitive transcription factors.

  4. A Comprehensive tRNA Genomic Survey Unravels the Evolutionary History of tRNA Arrays in Prokaryotes

    PubMed Central

    Tran, Tam T.T.; Belahbib, Hassiba; Bonnefoy, Violaine; Talla, Emmanuel

    2016-01-01

    Considering the importance of tRNAs in the translation machinery, scant attention has been paid to tRNA array units defined as genomic regions containing at least 20 tRNA genes with a minimal tRNA gene density of two tRNA genes per kilobase. Our analysis of Acidithiobacillus ferrivorans CF27 and Acidithiobacillus ferrooxidans ATCC 23270T genomes showed that both display a tRNA array unit with syntenic conservation which mainly contributed to the tRNA gene redundancy in these two organisms. Our investigations into the occurrence and distribution of tRNA array units revealed that 1) this tRNA organization is limited to few phyla and mainly found in Gram-positive bacteria; and 2) the presence of tRNA arrays favors the redundancy of tRNA genes, in particular those encoding the core tRNA isoacceptors. Finally, comparative array organization revealed that tRNA arrays were acquired through horizontal gene transfer (from Firmicutes or unknown donor), before being subjected to tRNA rearrangements, deletions, and duplications. In Bacilli, the most parsimonious evolutionary history involved two common ancestors and the acquisition of their arrays arose late in evolution, in the genera branches. Functional roles of the array units in organism lifestyle, selective genetic advantage and translation efficiency, as well as the evolutionary advantages of organisms harboring them were proposed. Our study offers new insight into the structural organization and evolution of tRNA arrays in prokaryotic organisms. PMID:26710853

  5. pH profile of the adsorption of nucleotides onto montmorillonite. II - Adsorption and desorption of 5-prime-AMP in iron-calcium montmorillonite systems

    NASA Technical Reports Server (NTRS)

    Banin, A.; Lawless, J. G.; Mazzurco, J.; Church, F. M.; Margulies, L.; Orenberg, J. B.

    1985-01-01

    The interaction of 5-prime-AMP with montmorillonite saturated with various ratios of two metals found ubiquitously on the surface of earth, that is, iron and calcium, is investigated. Adsorption and desorption of the nucleotide were studied in the pH range of 2-12 at three levels of addition: 0.080, 0.268 and 0.803 mmole 5-prime-AMP per gram of clay. Two desorption stages were employed - H2O wash and NaOH extraction (pH = 12.0). 5-prime-AMP was preferentially adsorbed on the Fe-containing clays relative to the Ca clay. The nucleotide was fully recovered by the two desorption stages, mostly by the NaOH extraction. The evidence at hand indicates that 5-prime-AMP reaction with clay is affected by electrostatic interactions involving both attraction and repulsion forces. Some specific adsorption, possibly the result of covalent bonding and complex formation with the adsorbed ion, cannot be ruled out for iron but does not appear to operate for calcium. Changes in pH cause varying degrees of attaction and repulsion of 5-prime-AMP and may have been operating on the primitive earth, leading to sequences of adsorption and release of this biomolecule.

  6. Interaction between fullerene halves Cn (n ≤ 40) and single wall carbon nanotube

    NASA Astrophysics Data System (ADS)

    Sharma, Amrish; Kaur, Sandeep; Mudahar, Isha

    2016-05-01

    We have investigated the structural and electronic properties of carbon nanotube with small fullerene halves Cn (n ≤ 40) which are covalently bonded to the side wall of an armchair single wall carbon nanotube (SWCNT) using first principle method based on density functional theory. The fullerene size results in weak bonding between fullerene halves and carbon nanotube (CNT). Further, it was found that the C-C bond distance that attaches the fullerene half and CNT is of the order of 1.60 Å. The calculated binding energies indicate the stability of the complexes formed. The HOMO-LUMO gaps and electron density of state plots points towards the metallicity of the complex formed. Our calculations on charge transfer reveal that very small amount of charge is transferred from CNT to fullerene halves.

  7. Compilation of tRNA sequences.

    PubMed

    Sprinzl, M; Grueter, F; Spelzhaus, A; Gauss, D H

    1980-01-11

    This compilation presents in a small space the tRNA sequences so far published. The numbering of tRNAPhe from yeast is used following the rules proposed by the participants of the Cold Spring Harbor Meeting on tRNA 1978 (1,2;Fig. 1). This numbering allows comparisons with the three dimensional structure of tRNAPhe. The secondary structure of tRNAs is indicated by specific underlining. In the primary structure a nucleoside followed by a nucleoside in brackets or a modification in brackets denotes that both types of nucleosides can occupy this position. Part of a sequence in brackets designates a piece of sequence not unambiguosly analyzed. Rare nucleosides are named according to the IUPACIUB rules (for complicated rare nucleosides and their identification see Table 1); those with lengthy names are given with the prefix x and specified in the footnotes. Footnotes are numbered according to the coordinates of the corresponding nucleoside and are indicated in the sequence by an asterisk. The references are restricted to the citation of the latest publication in those cases where several papers deal with one sequence. For additional information the reader is referred either to the original literature or to other tRNA sequence compilations (3-7). Mutant tRNAs are dealt with in a compilation by J. Celis (8). The compilers would welcome any information by the readers regarding missing material or erroneous presentation. On the basis of this numbering system computer printed compilations of tRNA sequences in a linear form and in cloverleaf form are in preparation. PMID:6986608

  8. Compilation of tRNA sequences.

    PubMed

    Gauss, D H; Grüter, F; Sprinzl, M

    1979-01-01

    This compilation presents in a small space the tRNA sequences so far published in order to enable rapid orientation and comparison. The numbering of tRNAPhe from yeast is used as has been done earlier (1) but following the rules proposed by the participants of the Cold Spring Harbor Meeting on tRNA 1978 (2) (Fig. 1). This numbering allows comparisons with the three dimensional structure of tRNAPhe, the only structure known from X-ray analysis. The secondary structure of tRNAs is indicated by specific underlining. In the primary structure a nucleoside followed by a nucleoside in brackets or a modification in brackets denotes that both types of nucleosides can occupy this position. Part of a sequence in brackets designates a piece of sequence not unambiguously analyzed. Rare nucleosides are named according to the IUPAC-IUB rules (for some more complicated rare nucleosides and their identification see Table 1); those with lengthy names are given with the prefix x and specified in the footnotes. Footnotes are numbered according to the coordinates of the corresponding nucleoside and are indicated in the sequence by an asterisk. The references are restricted to the citation of the latest publication in those cases where several papers deal with one sequence. For additional information the reader is referred either to the original literature or to other tRNA sequence compilations (3--7). Mutant tRNAs are dealt with in a separate compilation prepared by J. Celis (see below). The compilers would welcome any information by the readers regarding missing material or erroneous presentation. On the basis of this numbering system computer printed compilations of tRNA sequences in a linear form and in cloverleaf form are in preparation. PMID:424282

  9. Kinetic Analysis of tRNA Methylfransferases

    PubMed Central

    Hou, Ya-Ming; Masuda, Isao

    2016-01-01

    Transfer RNA (tRNA) molecules contain many chemical modifications that are introduced after transcription. A major form of these modifications is methyl transfer to bases and backbone groups, using S-adenosyl methionine (AdoMet) as the methyl donor. Each methylation confers a specific advantage to tRNA in structure or in function. A remarkable methylation is to the G37 base on the 3' side of the anticodon to generate m1G37-tRNA, which suppresses frameshift errors during protein synthesis and is therefore essential for cell growth in all three domains of life. This methylation is catalyzed by TrmD in bacteria and by Trm5 in eukaryotes and archaea. Although TrmD and Trm5 catalyze the same methylation reaction, kinetic analysis reveal that these two enzymes are unrelated to each other and are distinct in their reaction mechanism. This chapter summarizes the kinetic assays that are used to reveal the distinction between TrmD and Trm5. Three types of assays are described, the steady-state, the pre-steady-state, and the single turnover assays, which collectively provide the basis for mechanistic investigation of AdoMet-dependent methyl transfer reactions. PMID:26253967

  10. Linkage disequilibrium in the insulin gene region is related to the exact number of repeat units present at the 5{prime} flanking polymorphism

    SciTech Connect

    McGinnis, R.E.; Spielman, R.S.

    1994-09-01

    Tandem DNA repeat units (RUs) located 5{prime} to the insulin (INS) gene give rise to a {open_quotes}5{prime} flanking polymorphism{close_quotes} (5{prime}FP) with minisatellite alleles belonging to 3 size classes. The shortest or {open_quotes}class 1{close_quotes} alleles (mean length of {approximately}40 RUs) are associated with insulin-dependent diabetes mellitus (IDDM), and the 5{prime}FP is one of several INS region loci in strong linkage disequilibrium with IDDM. We have amplified class 1 alleles and have determined the exact number of RUs in individual class 1 alleles found in parents of 50 IDDM families. We also obtained INS region haplotypes by typing two loci near tyrosine hydroxylase (TH) and two loci near insulin-like growth factor II (IGF2). We obtained these results: (1) Class 1 alleles (n=101) were found at every integer length from 30 to 44 RUs, the lengths of smallest and largest class 1 alleles observed. The allele frequency distribution was trimodal with peaks at 31, 40 and 42 RUs; 18%, 34% and 48% of the alleles belonged to the three components, respectively. (2) Allelic variation at each flanking locus was highly associated with the exact number of RUs present at the 5{prime}FP. Our results suggest that creation of new 5{prime}FP or other minisatellite haplotypes may be {open_quotes}constrained{close_quotes} in that flanking alleles usually become associated with a new minisatellite length different by only one or two RUs. Furthermore, since many flanking alleles were associated with a single narrow range of class 1 integer lengths, determining exact RU length may aid in visualizing linkage disequilibrium and allelic associations involving other minisatellite loci.

  11. Stochastic context-free grammars for tRNA modeling.

    PubMed Central

    Sakakibara, Y; Brown, M; Hughey, R; Mian, I S; Sjölander, K; Underwood, R C; Haussler, D

    1994-01-01

    Stochastic context-free grammars (SCFGs) are applied to the problems of folding, aligning and modeling families of tRNA sequences. SCFGs capture the sequences' common primary and secondary structure and generalize the hidden Markov models (HMMs) used in related work on protein and DNA. Results show that after having been trained on as few as 20 tRNA sequences from only two tRNA subfamilies (mitochondrial and cytoplasmic), the model can discern general tRNA from similar-length RNA sequences of other kinds, can find secondary structure of new tRNA sequences, and can produce multiple alignments of large sets of tRNA sequences. Our results suggest potential improvements in the alignments of the D- and T-domains in some mitochondrial tRNAs that cannot be fit into the canonical secondary structure. PMID:7800507

  12. Structural Insights into tRNA Dynamics on the Ribosome

    PubMed Central

    Agirrezabala, Xabier; Valle, Mikel

    2015-01-01

    High-resolution structures at different stages, as well as biochemical, single molecule and computational approaches have highlighted the elasticity of tRNA molecules when bound to the ribosome. It is well acknowledged that the inherent structural flexibility of the tRNA lies at the heart of the protein synthesis process. Here, we review the recent advances and describe considerations that the conformational changes of the tRNA molecules offer about the mechanisms grounded in translation. PMID:25941930

  13. 40 CFR 761.306 - Sampling 1 meter square surfaces by random selection of halves.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Sampling 1 meter square surfaces by...(b)(3) § 761.306 Sampling 1 meter square surfaces by random selection of halves. (a) Divide each 1 meter square portion where it is necessary to collect a surface wipe test sample into two equal (or...

  14. 40 CFR 761.306 - Sampling 1 meter square surfaces by random selection of halves.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Sampling 1 meter square surfaces by...(b)(3) § 761.306 Sampling 1 meter square surfaces by random selection of halves. (a) Divide each 1 meter square portion where it is necessary to collect a surface wipe test sample into two equal (or...

  15. Analysis by illegitimate transcription of a mutation in the 5{prime} splice site in exon 8 of the PAH gene

    SciTech Connect

    Desviat, L.R.; Perez, B.; Ugarte, M.

    1994-09-01

    Up to now, 12 splice defects have been described within the PAH gene. Using PCR-SSCP and sequence analysis we have found a point mutation involving the last nucleotide in exon 8 (CAG/CAA). The G to A substitution does not alter the amino acid (Q204Q), but it may cause a splice defect, as it is included in the 5{prime} splice donor site, and the G at this position is highly conserved (80%) in all eukaryotic genes. We have analyzed by illegitimate transcription the PAH mRNA in lymphocytes of a patient bearing the mutation in a heterozygous fashion. After RT-PCR we observed once the appearance of an extra larger band, which could be due to the use of a cryptic splice site instead of the mutated one. Furthermore, sequencing of 6 clones of the band of expected size in the patient revealed that all had the normal sequence, in spite of the G to A substitution being found in the genomic DNA. In view of these results, we believe that the larger extra band represents the allele with the mutation which causes a highly unstable mis-spliced RNA. This splice defect could be, therefore, the disease causing mutation in the patient.

  16. Extracellular matrix and hormones transcriptionally regulate bovine. beta. -casein 5 prime sequences in stably transfected mouse mammary cells

    SciTech Connect

    Schmidhauser, C. Bissell, M.J. ); Myers, C.A.; Casperson, G.F. )

    1990-12-01

    Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-2D has been developed in which more than 35% of the cells express {beta}-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete {beta}-casein undirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine {beta}-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency. This regulation occurered primarily at the transcriptional level and was dependent on the length of the 5{prime} flanking region of the {beta}-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous {beta}-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

  17. Inactivation of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii by 2 prime -chloro-2 prime -deoxyuridine 5 prime -triphosphate: A 3 prime -2 prime hydrogen transfer during the formation of 3 prime -keto-2 prime -deoxyuridine 5 prime -triphosphate

    SciTech Connect

    Ashley, G.W.; Harris, G.; Stubbe, J. )

    1988-10-04

    The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2{prime}-chloro-2{prime}-deoxyuridine 5{prime}-triphosphate (C1UTP) into a mixture of 2{prime}-deoxyuridine triphosphate (dUTP) and the unstable product 3{prime}-keto-2{prime}-deoxyuridine triphosphate (3{prime}-keto-dUTP). This ketone can be trapped by reduction with NaBH{sub 4}, producing a 4:1 mixture of xylo-dUTP and dUTP. When (3{prime}-{sup 3}H)C1UTP is treated with enzyme in the presence of NaBH{sub 4}, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the {sup 3}H in C1UTP. Degradation of these isomeric nucleosides has established the location of the {sup 3}H in 3{prime}-keto-dUTP as predominantly 2{prime}(S). The xylo-dU had 98.6% of its label at the 2{prime}(S) position and 1.5% at 2{prime}(R). The isolated dU had 89.6% of its label at 2{prime}(S) and 1.4% at 2{prime}(R), with the remaining 9% label inferred to be at the 3{prime}-carbon, this resulting from the direct enzymic production of dUTP. These results are consistent with enzymic production of a 1:1,000 mixture of dUTP and 3{prime}-keto-dUTP, where the 3{prime}-hydrogen of C1UTP is retained at 3{prime} during production of dUTP and is transferred to 2{prime}(S) during production of 3{prime}-keto-dUTP. The implications of these results and the unique role of the cofactor adenosylcobalamin are discussed in terms of reductase being a model for the B{sub 12}-dependent rearrangement reactions.

  18. Genetic code from tRNA point of view.

    PubMed

    Chechetkin, V R

    2006-10-21

    The possible codon-anticodon pairings follow the standard genetic code, yet in a different mode. The corresponding rules for decoding sequence of the codons in mRNA with tRNA may be called "tRNA code". In this paper we analyse the mutational and translational stability of such tRNA code. Our approach is based on the model of "ambiguous intermediate" and on the study of underlying block structure and Eulerean graph technique. It is shown that the wobble rules and the reduced number of tRNA anticodons strongly affect the mutational and translational stability of the code. The selection of tRNA anticodons, besides the optimization of translation, also ensures the more reliable start and, to a lesser extent, the stop of translation. The attribution of tRNA anticodons to the groups [WWW, WWS, SWW, SWS] and [SSS, SSW, WSS, WSW] as well as [MMM, MMK, KMM, KMK] and [KKK, KKM, MKK, MKM] clearly correlates with class I and class II aminoacyl-tRNA synthetases and obeys the principle of the optimal coding in both cases. Both W-S and M-K groupings also refer to the encoding of amino acids with the large and small side-chain volumes, which may provide such an attribution. The higher variability of tRNA code agrees with the suggestions that the variations in an assignment of tRNA anticodons may serve as the driving force generating the different variants of the genetic code.

  19. Nucleotide sequence of a human tRNA gene heterocluster

    SciTech Connect

    Chang, Y.N.; Pirtle, I.L.; Pirtle, R.M.

    1986-05-01

    Leucine tRNA from bovine liver was used as a hybridization probe to screen a human gene library harbored in Charon-4A of bacteriophage lambda. The human DNA inserts from plaque-pure clones were characterized by restriction endonuclease mapping and Southern hybridization techniques, using both (3'-/sup 32/P)-labeled bovine liver leucine tRNA and total tRNA as hybridization probes. An 8-kb Hind III fragment of one of these ..gamma..-clones was subcloned into the Hind III site of pBR322. Subsequent fine restriction mapping and DNA sequence analysis of this plasmid DNA indicated the presence of four tRNA genes within the 8-kb DNA fragment. A leucine tRNA gene with an anticodon of AAG and a proline tRNA gene with an anticodon of AGG are in a 1.6-kb subfragment. A threonine tRNA gene with an anticodon of UGU and an as yet unidentified tRNA gene are located in a 1.1-kb subfragment. These two different subfragments are separated by 2.8 kb. The coding regions of the three sequenced genes contain characteristic internal split promoter sequences and do not have intervening sequences. The 3'-flanking region of these three genes have typical RNA polymerase III termination sites of at least four consecutive T residues.

  20. tRNA travels from the cytoplasm to organelles

    PubMed Central

    Rubio, Mary Anne T.; Hopper, Anita K.

    2011-01-01

    Transfer RNAs (tRNAs) encoded by the nuclear genome are surprisingly dynamic. Although tRNAs function in protein synthesis occurring on cytoplasmic ribosomes, tRNAs can transit from the cytoplasm to the nucleus and then again return to the cytoplasm by a process known as the tRNA retrograde process. Subsets of the cytoplasmic tRNAs are also imported into mitochondria and function in mitochondrial protein synthesis. The numbers of tRNA species that are imported into mitchondria differ among organisms, ranging from just a few to the entire set needed to decode mitochondrially encoded mRNAs. For some tRNAs, import is dependent on the mitochondrial protein import machinery, whereas the majority of tRNA mitochondrial import is independent of this machinery. Although cytoplasmic proteins and proteins located on the mitochondrial surface participating in the tRNA import process have been described for several organisms, the identity of these proteins differ among organisms. Likewise, the tRNA determinants required for mitochondrial import differ among tRNA species and organisms. Here, we present an overview and discuss the current state of knowledge regarding the mechanisms involved in the tRNA retrograde process and continue with an overview of tRNA import into mitochondria. Finally, we highlight areas of future research to understand the function and regulation of movement of tRNAs between the cytoplasm and organelles. PMID:21976284

  1. From Parts to Identity: Invariance and Sensitivity of Face Representations to Different Face Halves.

    PubMed

    Anzellotti, Stefano; Caramazza, Alfonso

    2016-05-01

    Recognizing the identity of a face is computationally challenging, because it requires distinguishing between similar images depicting different people, while recognizing even very different images depicting a same person. Previous human fMRI studies investigated representations of face identity in the presence of changes in viewpoint and in expression. Despite the importance of holistic processing for face recognition, an investigation of representations of face identity across different face parts is missing. To fill this gap, we investigated representations of face identity and their invariance across different face halves. Information about face identity with invariance across changes in the face half was individuated in the right anterior temporal lobe, indicating this region as the most plausible candidate brain area for the representation of face identity. In a complementary analysis, information distinguishing between different face halves was found to decline along the posterior to anterior axis in the ventral stream. PMID:25628344

  2. From Parts to Identity: Invariance and Sensitivity of Face Representations to Different Face Halves.

    PubMed

    Anzellotti, Stefano; Caramazza, Alfonso

    2016-05-01

    Recognizing the identity of a face is computationally challenging, because it requires distinguishing between similar images depicting different people, while recognizing even very different images depicting a same person. Previous human fMRI studies investigated representations of face identity in the presence of changes in viewpoint and in expression. Despite the importance of holistic processing for face recognition, an investigation of representations of face identity across different face parts is missing. To fill this gap, we investigated representations of face identity and their invariance across different face halves. Information about face identity with invariance across changes in the face half was individuated in the right anterior temporal lobe, indicating this region as the most plausible candidate brain area for the representation of face identity. In a complementary analysis, information distinguishing between different face halves was found to decline along the posterior to anterior axis in the ventral stream.

  3. Extensive tRNA gene changes in synthetic Brassica napus.

    PubMed

    Wei, Lijuan; An, Zeshan; Mason, Annaliese S; Xiao, Meili; Guo, Ying; Yin, Jiaming; Li, Jiana; Fu, Donghui

    2014-01-01

    Allopolyploidization, where two species come together to form a new species, plays a major role in speciation and genome evolution. Transfer RNAs (abbreviated tRNA) are typically 73-94 nucleotides in length, and are indispensable in protein synthesis, transferring amino acids to the cell protein synthesis machinery (ribosome). To date, the regularity and function of tRNA gene sequence variation during the process of allopolyploidization have not been well understood. In this study, the inter-tRNA gene corresponding to tRNA amplification polymorphism method was used to detect changes in tRNA gene sequences in the progeny of interspecific hybrids between Brassica rapa and B. oleracea, mimicking the original B. napus (canola) species formation event. Cluster analysis showed that tRNA gene variation during allopolyploidization did not appear to have a genotypic basis. Significant variation occurred in the early generations of synthetic B. napus (F1 and F2 generations), but fewer alterations were observed in the later generation (F3). The variation-prone tRNA genes tended to be located in AT-rich regions. BlastN analysis of novel tRNA gene variants against a Brassica genome sequence database showed that the variation of these tRNA-gene-associated sequences in allopolyploidization might result in variation of gene structure and function, e.g., metabolic process and transport.

  4. Diversity in mechanism and function of tRNA methyltransferases

    PubMed Central

    Swinehart, William E; Jackman, Jane E

    2015-01-01

    tRNA molecules undergo extensive post-transcriptional processing to generate the mature functional tRNA species that are essential for translation in all organisms. These processing steps include the introduction of numerous specific chemical modifications to nucleotide bases and sugars; among these modifications, methylation reactions are by far the most abundant. The tRNA methyltransferases comprise a diverse enzyme superfamily, including members of multiple structural classes that appear to have arisen independently during evolution. Even among closely related family members, examples of unusual substrate specificity and chemistry have been observed. Here we review recent advances in tRNA methyltransferase mechanism and function with a particular emphasis on discoveries of alternative substrate specificities and chemistry associated with some methyltransferases. Although the molecular function for a specific tRNA methylation may not always be clear, mutations in tRNA methyltransferases have been increasingly associated with human disease. The impact of tRNA methylation on human biology is also discussed. PMID:25626150

  5. Handling tRNA introns, archaeal way and eukaryotic way

    PubMed Central

    Yoshihisa, Tohru

    2014-01-01

    Introns are found in various tRNA genes in all the three kingdoms of life. Especially, archaeal and eukaryotic genomes are good sources of tRNA introns that are removed by proteinaceous splicing machinery. Most intron-containing tRNA genes both in archaea and eukaryotes possess an intron at a so-called canonical position, one nucleotide 3′ to their anticodon, while recent bioinformatics have revealed unusual types of tRNA introns and their derivatives especially in archaeal genomes. Gain and loss of tRNA introns during various stages of evolution are obvious both in archaea and eukaryotes from analyses of comparative genomics. The splicing of tRNA molecules has been studied extensively from biochemical and cell biological points of view, and such analyses of eukaryotic systems provided interesting findings in the past years. Here, I summarize recent progresses in the analyses of tRNA introns and the splicing process, and try to clarify new and old questions to be solved in the next stages. PMID:25071838

  6. RNA Ligation and the Origin of tRNA

    NASA Astrophysics Data System (ADS)

    Nagaswamy, Uma; Fox, George E.

    2003-04-01

    A straightforward origin of transfer RNA, (tRNA), is difficult to envision because of the apparently complex idiosyncratic interaction between the D-loop and T-loop. Recently, multiple examples of the T-loop structural motif have been identified in ribosomal RNA. These examples show that the long-range interactions between the T-loop and D-loops seen in tRNA are not an essential part of the motif but rather are facilitated by it. Thus, the core T-loop structure could already have existed in a small RNA prior to the emergence of the tRNA. The tRNA might then have arisen by expansion of an RNA that carried the motif. With this idea in mind, Di Giulio's earlier hypothesis that tRNA evolved by a simple duplication or ligation of a minihelix RNA was re-examined. It is shown that an essentially modern tRNA structure can in fact be generated by the ligation of two 38-nucleotide RNA minihelices of appropriate sequence. Although rare, such sequences occur with sufficient frequency, (1 in 3 × 107), that they could be found in a standard in vitro RNA selection experiment. The results demonstrate that a series of RNA duplications, as previously proposed, can in principal account for the origin of tRNA. More generally, the results point out that RNA ligation can be a powerful driving force for increased complexity in the RNA World.

  7. Insertion of part of an intron into the 5[prime] untranslated region of a Caenorhabditis elegans gene converts it into a trans-spliced gene

    SciTech Connect

    Conrad, R.; Thomas, J.; Spieth, J.; Blumenthal, T. )

    1991-04-01

    In nematodes, the RNA products of some genes are trans-spliced to a 22-nucleotide spliced leader (SL), while the RNA products of other genes are not. In Caenorhabditis elegans, there are two SLs, Sl1 and SL2, donated by two distinct small nuclear ribonucleoprotein particles in a process functionally quite similar to nuclear intron removal. The authors demonstrate here that it is possible to convert a non-trans-spliced gene into a trans-spliced gene by placement of an intron missing only the 5[prime] splice site into the 5[prime] untranslated region. Stable transgenic strains were isolated expressing a gene in which 69 nucleotides of a vit-5 intron, including the 3[prime] splice site, were inserted into the 5[prime] untranslated region of a vit-2/vit-6 fusion gene. The RNA product of this gene was examined by primer extension and PCR amplification. Although the vit-2/vit-6 transgene product is not normally trans-spliced, the majority of transcripts from this altered gene were trans-spliced to SL1. They termed the region of a trans-spliced mRNA precursor between the 5[prime] end and the first 3[prime] splice site an 'outrun'. The results suggest that if a transcript begins with intronlike sequence followed by a 3[prime] splice site, this alone may constitute an outrun and be sufficient to demarcate a transcript as a trans-splice acceptor. These findings leave open the possibility that specific sequences are required to increase the efficiency of trans-splicing.

  8. Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5 prime -triphosphate

    SciTech Connect

    Vollmer, S.H.; Colman, R.F. )

    1990-03-13

    The affinity label 8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5{prime}-triphosphate (8-BDB-TA-5{prime}-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K{sup +}, and Mn{sup 2+} and only 1 mol of reagent/mol of subunit is incorporated. The authors have now identified the resultant modified residues. After reaction with 8-BDB-TA-5{prime}-TP at pH 7.0, modified enzyme was incubated with ({sup 3}H)NaBH{sub 4} to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5{prime}-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn{sup 162}-Ile-Cys-Lys{sup 165} and Cys{sup 151}-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys{sup 161}, with a smaller amount of Asn{sup 43}-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg{sup 55}. Reaction in the presence of the protectants phosphoenolpyruvate, K{sup +}, and Mn{sup 2+} yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys{sup 151} and Cys{sup 48}.

  9. The La protein functions redundantly with tRNA modification enzymes to ensure tRNA structural stability.

    PubMed

    Copela, Laura A; Chakshusmathi, Ghadiyaram; Sherrer, R Lynn; Wolin, Sandra L

    2006-04-01

    Although the La protein stabilizes nascent pre-tRNAs from nucleases, influences the pathway of pre-tRNA maturation, and assists correct folding of certain pre-tRNAs, it is dispensable for growth in both budding and fission yeast. Here we show that the Saccharomyces cerevisiae La shares functional redundancy with both tRNA modification enzymes and other proteins that contact tRNAs during their biogenesis. La is important for growth in the presence of mutations in either the arginyl tRNA synthetase or the tRNA modification enzyme Trm1p. In addition, two pseudouridine synthases, PUS3 and PUS4, are important for growth in strains carrying a mutation in tRNA(Arg)(CCG) and are essential when La is deleted in these strains. Depletion of Pus3p results in accumulation of the aminoacylated mutant tRNA(Arg)(CCG) in nuclei, while depletion of Pus4p results in decreased stability of the mutant tRNA. Interestingly, the degradation of mutant unstable forms of tRNA(Arg)(CCG) does not require the Trf4p poly(A) polymerase, suggesting that yeast cells possess multiple pathways for tRNA decay. These data demonstrate that La functions redundantly with both tRNA modifications and proteins that associate with tRNAs to achieve tRNA structural stability and efficient biogenesis.

  10. GA Enhanced a-Amylase Synthesis in Halved Grains of Barley (Hordeum vulgare): A Simple Laboratory Demonstration

    ERIC Educational Resources Information Center

    Freeland, P. W.

    1972-01-01

    A laboratory demonstration is suggested for the formation of a-amylase enzyme in halved grains of barley. Data presented in the article provide some information of the pattern of a- and b-amylase activity during germination. (PS)

  11. Stable tRNA precursors in HeLa cells.

    PubMed Central

    Harada, F; Matsubara, M; Kato, N

    1984-01-01

    Two tRNA precursors were isolated from 32P-labeled or unlabeled HeLa cells by two dimensional polyacrylamide gel electrophoresis, and were sequenced. These were the precursors of tRNAMet and tRNALeu, and both contained four extra nucleotides including 5'-triphosphates at their 5'-end and nine extra nucleotides including oligo U at their 3'-end. These RNAs are the first naturally occurring tRNA precursors from higher eukaryotes whose sequences have been determined. In these molecules, several modified nucleosides such as m2G, t6A and ac4C in mature tRNAs were undermodified. Two additional hydrogen bonds were formed in the clover leaf structures of these tRNA precursors. These extra hydrogen bonds may be responsible for the stabilities of these tRNA precursors. Images PMID:6514577

  12. Origins and Early Evolution of the tRNA Molecule

    PubMed Central

    Tamura, Koji

    2015-01-01

    Modern transfer RNAs (tRNAs) are composed of ~76 nucleotides and play an important role as “adaptor” molecules that mediate the translation of information from messenger RNAs (mRNAs). Many studies suggest that the contemporary full-length tRNA was formed by the ligation of half-sized hairpin-like RNAs. A minihelix (a coaxial stack of the acceptor stem on the T-stem of tRNA) can function both in aminoacylation by aminoacyl tRNA synthetases and in peptide bond formation on the ribosome, indicating that it may be a vestige of the ancestral tRNA. The universal CCA-3′ terminus of tRNA is also a typical characteristic of the molecule. “Why CCA?” is the fundamental unanswered question, but several findings give a comprehensive picture of its origin. Here, the origins and early evolution of tRNA are discussed in terms of various perspectives, including nucleotide ligation, chiral selectivity of amino acids, genetic code evolution, and the organization of the ribosomal peptidyl transferase center (PTC). The proto-tRNA molecules may have evolved not only as adaptors but also as contributors to the composition of the ribosome. PMID:26633518

  13. Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

    PubMed Central

    Capone, J P; Sharp, P A; RajBhandary, U L

    1985-01-01

    Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2990894

  14. Tissue-specific methylation of individual CpG dinucleotides in the 5{prime} upstream region of the mouse catalase gene (Cas-1)

    SciTech Connect

    Pillay, I.L.; Singh, S.M.

    1994-09-01

    The intracellular antioxidant enzyme, catalase, is encoded by a gene whose level of expression in different organisms, including humans, varies with tissue-type. The {open_quotes}TATA-less{close_quotes} 5{prime} upstream region of the catalase gene, in mice and humans, contains a CpG island. Such CG-rich regions are target sites for cytosine methylation and have been implicated in tissue-specific gene expression. However, the methylation status of individual CpG dinucleotides and their significance in gene expression has not been established. A 275 bp fragment within the 5{prime} region of Cas-1 was evaluated for CpG methylation. HpaII digestion of genomic DNA, followed by polymerase chain reaction amplification (HpaII-PCR), suggests that at least one of three CCGG is not methylated in nine different somatic tissues that express this enzyme at various levels. In contrast, all three CCGG sites are methylated in DNA from sperm and spleen. Further examination of the methylation specificity of individual CCGG sites was conducted using sodium bisulfite modification of genomic DNA followed by HPaII-PCR. Sodium bisulfite modifies non-methylated cytosines to uracils, changing a CG to a TG dinucleotide. This nucleotide substitution eliminates HpaII sites and allows the methylation status of each of the CCGG sites to be assessed. The ability to discern the number and combination of methylated sites within the 5{prime} region of a gene permits the determination of a possible correlation between differential methylation patterns and temporal/spatial gene regulation. Analysis of differential methylation, using the mouse catalase gene as a model, provides further insight into CpG methylation as one mechanism of mammalian gene regulation.

  15. A new family of retroviral long terminal repeat elements in the human genome identified by their homologies to an element 5{prime} to the spider monkey haptoglobin gene

    SciTech Connect

    Erickson, L.M.; Maeda, N.

    1995-06-10

    A new family of retroviral long terminal repeats that we name Spm-LTR has been identified as a result of DNA sequence comparisons between the entire Gen-Bank databank and an element, SPHP, located 5{prime} to the haptoglobin gene of spider monkeys. The 18 human Spm-LTR sequences so identified fall into three subtypes. There is no sequence similarity between Spm-LTR elements and any endogenous retroviral LTR sequences previously reported except for general features that define LTRs. However, a previously described repeated sequence (MER-4) forms a portion of the Spm-LTR sequence. 13 refs., 1 fig., 1 tab.

  16. Early molecular response in chronic myeloid leukemia and halving time: Latest evidences.

    PubMed

    Breccia, Massimo; Molica, Matteo; Colafigli, Gioia; Massaro, Fulvio; Alimena, Giuliana

    2016-09-01

    Achieving a BCR-ABL/ABL ratio <10% at 3 months has become an important goal of treatment for chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. Several evidences showed that this early molecular response (EMR) is associated with positive long-term outcome in terms of overall survival and progression-free survival, but a consensus has not been reached when this goal is not achieved. European LeukemiaNet recommendations defined patients as treatment failure only after the 6- month time point. Not all patients that lack EMR have similar outcome and it became important to identify patients before this time point of 3 months. Several groups introduced the concept of "halving time" or "velocity of ratio reduction" that could anticipate the possibility to recognize patients deserving a switch to another treatment. Aim of this review is to summarize all evidences reported on the significance of EMR and how this evaluation changed our perspectives and modified our therapeutic strategies.

  17. Reverse Translocation of tRNA in the Ribosome

    PubMed Central

    Shoji, Shinichiro; Walker, Sarah E.; Fredrick, Kurt

    2009-01-01

    Summary A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have important implications for the energetics of translocation. PMID:17189194

  18. Synthesis and characterization of ( sup 3 H)-5 prime azido-N-1-naphthylphthlamic acid, a photolabile N-1-naphthylphthalamic acid analog

    SciTech Connect

    Voet, J.G.; Dodge, B.; Harris, K.; Jacobs, M.; Larkin, L.; Bader, S.; Schnitzler, G.; Sutherland, J. )

    1990-05-01

    The NPA (N-1-naphthylphthalamic acid) receptor is an important protein involved in the regulation of transport of indole-3-acetic acid (IAA). In our attempt to isolate and characterize this protein we have previously synthesized and characterized a photolabile analog of NPA, 5{prime}-azido-NPA (Az-NPA) and shown it to be a competitor of NPA for binding sites on the NPA receptor as well as an inhibitor of auxin transport. We have now synthesized and characterized ({sup 3}H)-Az-NPA. The precursor, 2,3,4,5-Br-5{prime}-amino-NPA was dehydrohalogenated with tritium gas by Research Products International. The amino group was converted to an azido group and the product purified by HPLC. ({sup 3}H)-Az-NPA was found to be photolabile and to co-chromatograph with our synthetic unlabeled Az-NPA. Furthermore, the tritiated material was found to bind to zucchini hypocotyl cell membranes in a manner competitive with NPA as well as unlabeled Az-NPA. Photolysis of zucchini phase-partitioned plasma membranes in the presence of ({sup 3}H)-Az-NPA resulted in covalent association of tritium with the membranes. Much of this covalent association could be prevented by prior treatment of the membranes with excess NPA.

  19. EXISTENCE OF RULE OF HALVES IN HYPERTENSION: AN EXPLORATORY ANALYSIS IN AN INDIAN VILLAGE

    PubMed Central

    Faizi, Nafis; Ahmad, Anees; Khalique, Najam; Shah, Mohammad Salman; Khan, Mohammad Shibly; Maroof, Mohd

    2016-01-01

    Introduction: India is a country in transition, the population is graying and the non communicable diseases are rising. In the rural areas of India, the detection of hypertension is poor because of limited healthcare facilities and poor awareness among the people. In one such village, Mirzapur, adopted by the Aligarh Muslim University, there is a planned project to control hypertension in the villages through some innovative approaches. This study was the assessment phase of this project for mass management of hypertension to steer and guide the next phase of the project. Aim: The main objectives of this study were: to determine the prevalence of hypertension in residents ≥ 40 years in Mirzapur village, Aligarh, and, to assess the presence of rule of halves in hypertension Results and Discussion: The present study in residents more than 40 years of age in the Mirzapur village in Aligarh found that the prevalence of hypertension in the study population was 41.9%, with a higher prevalence in older age groups. The mean blood pressure of the study population was found to be 100.03±13.17 mm Hg. The high prevalence reported in the present study reflects and reaffirms the increasing trend of hypertension in not only the urban, but also rural India, at least in the older age group. The problem of hypertension, due to its silent and asymptomatic nature, frequently depicts a rule of halves in places with weaker health system and an equally weaker health awareness and information among populations and the same is true for this village. Conclusion: There is an urgent need to conduct similar researches in other adopted villages of the country for the sake of inclusive development to find the exact burden of this silent and asymptomatic killer. More importantly, there is a need to find innovative solutions to combat the problem of hypertension detection and management. PMID:27147912

  20. The tRNA Recognition Mechanism of Folate/FAD-dependent tRNA Methyltransferase (TrmFO)*

    PubMed Central

    Yamagami, Ryota; Yamashita, Koki; Nishimasu, Hiroshi; Tomikawa, Chie; Ochi, Anna; Iwashita, Chikako; Hirata, Akira; Ishitani, Ryuichiro; Nureki, Osamu; Hori, Hiroyuki

    2012-01-01

    The conserved U54 in tRNA is often modified to 5-methyluridine (m5U) and forms a reverse Hoogsteen base pair with A58 that stabilizes the L-shaped tRNA structure. In Gram-positive and some Gram-negative eubacteria, m5U54 is produced by folate/FAD-dependent tRNA (m5U54) methyltransferase (TrmFO). TrmFO utilizes N5,N10-methylenetetrahydrofolate (CH2THF) as a methyl donor. We previously reported an in vitro TrmFO assay system, in which unstable [14C]CH2THF was supplied from [14C]serine and tetrahydrofolate by serine hydroxymethyltransferase. In the current study, we have improved the TrmFO assay system by optimization of enzyme and substrate concentrations and introduction of a filter assay system. Using this assay, we have focused on the tRNA recognition mechanism of TrmFO. 42 tRNA mutant variants were prepared, and experiments with truncated tRNA and microhelix RNAs revealed that the minimum requirement of TrmFO exists in the T-arm structure. The positive determinants for TrmFO were found to be the U54U55C56 sequence and G53-C61 base pair. The gel mobility shift assay and fluorescence quenching showed that the affinity of TrmFO for tRNA in the initial binding process is weak. The inhibition experiments showed that the methylated tRNA is released before the structural change process. Furthermore, we found that A38 prevents incorrect methylation of U32 in the anticodon loop. Moreover, the m1A58 modification clearly accelerates the TrmFO reaction, suggesting a synergistic effect of the m5U54, m1A58, and s2U54 modifications on m5s2U54 formation in Thermus thermophilus cells. The docking model of TrmFO and the T-arm showed that the G53-C61 base pair is not able to directly contact the enzyme. PMID:23095745

  1. Linkage disequilibrium between polymorphisms at the 5{prime} untranslated region and intron 5 (Dde I) of the antithrombin III (ATIII) gene in the Chinese

    SciTech Connect

    Tay, J.S.H.; Liu, Y.; Low, P.S.

    1994-09-01

    A length polymorphism at the 5{prime} untranslated region of exon 1 and an RFLP (Dde I) in intron 5 (nt 160) of the ATIII gene were amplified by polymerase chain reaction with primers of published sequences. DNA fragments were size-fractionated by agarose gel electrophoresis (3% NuSieve and 1% Seakem GTG) and photographed over a UV transilluminator. A strong linkage disequilibrium was observed between these two polymorphisms of the ATIII gene in the Chinese ({chi}{sup 2} = 63.7; {triangle} 0.42, P < 0.001). The estimated frequencies of the three haplotypes were found to be 0.37 for SD+, 0.40 for LD+ and 0.23 for LD-.

  2. Early molecular response in chronic myeloid leukemia and halving time: Latest evidences.

    PubMed

    Breccia, Massimo; Molica, Matteo; Colafigli, Gioia; Massaro, Fulvio; Alimena, Giuliana

    2016-09-01

    Achieving a BCR-ABL/ABL ratio <10% at 3 months has become an important goal of treatment for chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. Several evidences showed that this early molecular response (EMR) is associated with positive long-term outcome in terms of overall survival and progression-free survival, but a consensus has not been reached when this goal is not achieved. European LeukemiaNet recommendations defined patients as treatment failure only after the 6- month time point. Not all patients that lack EMR have similar outcome and it became important to identify patients before this time point of 3 months. Several groups introduced the concept of "halving time" or "velocity of ratio reduction" that could anticipate the possibility to recognize patients deserving a switch to another treatment. Aim of this review is to summarize all evidences reported on the significance of EMR and how this evaluation changed our perspectives and modified our therapeutic strategies. PMID:27442893

  3. Dynamics of tRNA translocation, mRNA translocation and tRNA dissociation during ribosome translation through mRNA secondary structures.

    PubMed

    Xie, Ping

    2014-07-01

    The ribosome can translate through the duplex region or secondary structure of mRNA. Recent single-molecule experimental data showed that downstream mRNA secondary structures have more sensitive effects on deacylated tRNA dissociation from the E site than on tRNA translocation in the 50S subunit. However, it is unclear how the downstream mRNA secondary structure can affect the tRNA dissociation from the E site, which is distant from the secondary structure. Here, based on our proposed ribosomal translocation model, we theoretically study the dynamics of tRNA translocation in the 50S subunit, mRNA translocation and tRNA dissociation, giving quantitative explanations of the single-molecule experimental data. It is shown that the effect of the downstream mRNA secondary structure on tRNA dissociation is via the effect on mRNA translocation, while the mRNA secondary structure has no effect on the rate of deacylated tRNA dissociation from the posttranslocation state. The slow mRNA translocation, which results in slow tRNA dissociation, derives from the occurrence of the futile transition, which is induced by the energy barrier from base pair unwinding to resist the forward translocation. The reduced translation rate through the mRNA secondary structure is induced by the slow mRNA translocation rather than the slow tRNA dissociation.

  4. Identification of the determinants of tRNA function and susceptibility to rapid tRNA decay by high-throughput in vivo analysis.

    PubMed

    Guy, Michael P; Young, David L; Payea, Matthew J; Zhang, Xiaoju; Kon, Yoshiko; Dean, Kimberly M; Grayhack, Elizabeth J; Mathews, David H; Fields, Stanley; Phizicky, Eric M

    2014-08-01

    Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an ochre mutation, fluorescence-activated cell sorting of a library of SUP4oc mutant yeast strains, and deep sequencing, we scored 25,491 variants. Unexpectedly, SUP4oc tolerates numerous sequence variations, accommodates slippage in tertiary and secondary interactions, and exhibits genetic interactions that suggest an alternative functional tRNA conformation. Furthermore, we used this methodology to define tRNA variants subject to rapid tRNA decay (RTD). Even though RTD normally degrades tRNAs with exposed 5' ends, mutations that sensitize SUP4oc to RTD were found to be located throughout the sequence, including the anti-codon stem. Thus, the integrity of the entire tRNA molecule is under surveillance by cellular quality control machinery. This approach to assess activity at high throughput is widely applicable to many problems in tRNA biology. PMID:25085423

  5. Translocation and rotation of tRNA during template-independent RNA polymerization by tRNA nucleotidyltransferase.

    PubMed

    Yamashita, Seisuke; Takeshita, Daijiro; Tomita, Kozo

    2014-02-01

    The 3'-terminal CCA (CCA-3' at positions 74-76) of tRNA is synthesized by CCA-adding enzyme using CTP and ATP as substrates, without a nucleic acid template. In Aquifex aeolicus, CC-adding and A-adding enzymes collaboratively synthesize the CCA-3'. The mechanism of CCA-3' synthesis by these two enzymes remained obscure. We now present crystal structures representing CC addition onto tRNA by A. aeolicus CC-adding enzyme. After C₇₄ addition in an enclosed active pocket and pyrophosphate release, the tRNA translocates and rotates relative to the enzyme, and C₇₅ addition occurs in the same active pocket as C₇₄ addition. At both the C₇₄-adding and C₇₅-adding stages, CTP is selected by Watson-Crick-like hydrogen bonds between the cytosine of CTP and conserved Asp and Arg residues in the pocket. After C₇₄C₇₅ addition and pyrophosphate release, the tRNA translocates further and drops off the enzyme, and the CC-adding enzyme terminates RNA polymerization.

  6. Data on true tRNA diversity among uncultured and bacterial strains

    PubMed Central

    Rekadwad, Bhagwan N.; Khobragade, Chandrahasya N.

    2016-01-01

    Complete genome sequences of two uncultured archaea (BX649197 and CR937008) and 10 uncultured bacteria (AC160099, FP245538-FP245540, FP312972, FP312974-75, FP312977, FP312985 and NZ_JPJG01000067) were used for creation of digital data of tRNA. tRNAscan-SE and ENDMEMO GC calculating tools were used for detection of tRNA, drawing their structures and calculation of GC percent. Seven archaeal and 48 bacterial tRNA were detected from above 12 sequences. Four archaeal and 30 bacterial tRNA showed cove score more than 20% are called as true tRNA. Three tRNA of uncultured bacteria (AC160099) has the presence of the variable loop. The tRNA of FP245540, FP245575, FP245577 and FP245585 has one variable loop each. The true tRNA of archaea were Alanine, Arginine and Cysteine-type tRNA, while the majority of bacteria true tRNA classified as Alanine, Glutamic acid, Isoleucine, Leucine, Methionine, Phenylalanine, Proline and Valine-type tRNA with cove score ranged from 70% to 97.15%. Archaeal and bacterial have GC content approximately 43% and 34.7–63.3% respectively. Archaeal tRNA has 60.4–64.2% GC content. Similarly, bacterial tRNA contributed 49.3–66.3% GC content to the total GC content. This generated data is useful for studies on diversity of tRNA among prokaryotes. PMID:27222849

  7. Factor for inversion stimulation-dependent growth rate regulation of individual tRNA species in Escherichia coli.

    PubMed

    Nilsson, L; Emilsson, V

    1994-04-01

    We have studied the involvement of the factor for inversion stimulation (FIS) in the growth rate-dependent expression of the arginine, leucine, and methionine acceptor tRNA species. The concentration of individual tRNA species relative to 16 S rRNA was determined by blot hybridization using RNA preparations from bacteria with the fis gene deleted and from isogenic wild type bacteria. The RNA preparations were obtained from bacteria growing under steady state conditions in different media. The levels of tRNA(1Leu), tRNA(2Arg), tRNA(4Arg), and tRNA(5Arg decreased in the fis bacteria, relative to the wild type. The difference in levels increased with increasing growth rate. Surprisingly, tRNA(3Leu), tRNA(rMet), and tRNA(eMet) showed the opposite response, with an increase of the tRNA/16 S ratio in the fis bacteria. The tRNA(2Leu, tRNA(4Leu), tRNA(5Leu), and tRNA(3 Arg) had unaffected tRNA/16 S ratios in fis cells. We conclude that FIS, directly or indirectly, is involved in growth rate regulation of some tRNA species and that it affects the composition of the cellular tRNA pool.

  8. The Selenocysteine tRNA STAF-Binding Region is Essential for Adequate Selenocysteine tRNA Status, Selenoprotein Expression and Early Age Survival of Mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    STAF is a transcription activating factor for a number of RNA Pol III-and RNA Pol II-dependent genes including the selenocysteine (Sec) tRNA gene. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined in an invivo model. Heterozygous inactivation of the Staf gen...

  9. Diversity of human tRNA genes from the 1000-genomes project

    PubMed Central

    Parisien, Marc; Wang, Xiaoyun; Pan, Tao

    2013-01-01

    The sequence diversity of individual human genomes has been extensively analyzed for variations and phenotypic implications for mRNA, miRNA, and long non-coding RNA genes. TRNA (tRNA) also exhibits large sequence diversity in the human genome, but tRNA gene sequence variation and potential functional implications in individual human genomes have not been investigated. Here we capitalize on the sequencing data from the 1000-genomes project to examine the diversity of tRNA genes in the human population. Previous analysis of the reference human genome indicated an unexpected large number of diverse tRNA genes beyond the necessity of translation, suggesting that some tRNA transcripts may perform non-canonical functions. We found 24 new tRNA sequences in > 1% and 76 new tRNA sequences in > 0.2% of all individuals, indicating that tRNA genes are also subject to evolutionary changes in the human population. Unexpectedly, two abundant new tRNA genes contain base-pair mismatches in the anticodon stem. We experimentally determined that these two new tRNAs have altered structures in vitro; however, one new tRNA is not aminoacylated but extremely stable in HeLa cells, suggesting that this new tRNA can be used for non-canonical function. Our results show that at the scale of human population, tRNA genes are more diverse than conventionally understood, and some new tRNAs may perform non-canonical, extra-translational functions that may be linked to human health and disease. PMID:24448271

  10. Diversity of human tRNA genes from the 1000-genomes project.

    PubMed

    Parisien, Marc; Wang, Xiaoyun; Pan, Tao

    2013-12-01

    The sequence diversity of individual human genomes has been extensively analyzed for variations and phenotypic implications for mRNA, miRNA, and long non-coding RNA genes. TRNA (tRNA) also exhibits large sequence diversity in the human genome, but tRNA gene sequence variation and potential functional implications in individual human genomes have not been investigated. Here we capitalize on the sequencing data from the 1000-genomes project to examine the diversity of tRNA genes in the human population. Previous analysis of the reference human genome indicated an unexpected large number of diverse tRNA genes beyond the necessity of translation, suggesting that some tRNA transcripts may perform non-canonical functions. We found 24 new tRNA sequences in>1% and 76 new tRNA sequences in>0.2% of all individuals, indicating that tRNA genes are also subject to evolutionary changes in the human population. Unexpectedly, two abundant new tRNA genes contain base-pair mismatches in the anticodon stem. We experimentally determined that these two new tRNAs have altered structures in vitro; however, one new tRNA is not aminoacylated but extremely stable in HeLa cells, suggesting that this new tRNA can be used for non-canonical function. Our results show that at the scale of human population, tRNA genes are more diverse than conventionally understood, and some new tRNAs may perform non-canonical, extra-translational functions that may be linked to human health and disease.

  11. The chicken FMR1 gene is highly conserved with a CCT 5{prime} - untranslated repeat and encodes an RNA-binding protein

    SciTech Connect

    Price, D.K.; Zhang, F.; Ashley, C.T. Jr.; Warren, S.T.

    1996-01-01

    The transcriptional silencing of the human gene, fragile X metal retardation 1 (FMR1), is due to abnormal methylation in response to an expanded 5{prime}-untranslated CGG trinucleotide repeat and accounts for most cases of fragile X syndrome, a frequent inherited form of metal retardation. Although the encoded fragile X mental retardation protein (FMRP) is known to have properties of a RNA-binding protein, the precise function of FMRP remains to be elucidated. We report the cloning of the chicken homolog of FMR1 and show strong evolutionary conservation, with nucleotide and amino acid identities of 85 and 92%, respectively, between chicken and human. In place of the mammalian CGG trinucleotide repeat, a 99-nt tripartite repetitive element containing a CCT trinucleotide repeat flanked on both sides by dinucleotide repeats was identified. Blocks of highly conserved 3{prime}-untranslated sequence were also found. Within the coding region, two copies each of the highly conserved K homology motif and the Arg-Gly-Gly (RGG) box motif, both ribonucleotide particle family domains implicated in RNA binding, were identified. Chicken FMRP was found to bind RNA in vitro, and this activity correlated with the presence of the carboxy-terminal portion of the protein that includes the RGG motifs. 49 refs., 7 figs.

  12. Increased inosine 5{prime}-monophosphate dehydrogenase gene expression in replicating cells: A response to growth factors, not to changes in cell cycle parameters

    SciTech Connect

    Tsutani, Hiroshi; Collart, F.R.; Glesne, D.A.; Huberman, E. |

    1997-07-01

    The authors have analyzed levels of inosine 5{prime}-monophosphate dehydrogenase (IMPDH; E.C. 1.1.1.205) type II mRNA levels in a human melanoma cell line, SK-MEL-131, and a Chinese hamster ovary cell line synchronously progressing through the cell cycle following treatment with aphidicolin. Following release from the aphidicolin block at the G{sub 1}-S phase boundary, the type II IMPDH gene was found to be constitutively expressed at a similar level during all stages of the cell cycle. To analyze growth regulation, as opposed to cell cycle regulation, stable SK-MEL-131 transfectants that express a type II IMPDH-promoted heterologous construct were assayed following deprivation of serum growth factors and after restimulation with fresh serum. Serum deprivation resulted in down-regulation of both steady state type II IMPDH mRNA levels and promoter activity, while restimulation with serum resulted in up-regulation of these parameters. These findings support the conclusion that the increase in IMPDH type II gene expression in replicating cells is mainly due to growth factor regulation rather than changes in cell cycle parameters and that this regulation is mediated primarily by a transcriptional mechanism. The increased level of IMPDH expression and activity found in many tumors may therefore also be due to a transcriptionally mediated response to growth factors.

  13. Mistranslation and its control by tRNA synthetases

    PubMed Central

    Schimmel, Paul

    2011-01-01

    Aminoacyl tRNA synthetases are ancient proteins that interpret the genetic material in all life forms. They are thought to have appeared during the transition from the RNA world to the theatre of proteins. During translation, they establish the rules of the genetic code, whereby each amino acid is attached to a tRNA that is cognate to the amino acid. Mistranslation occurs when an amino acid is attached to the wrong tRNA and subsequently is misplaced in a nascent protein. Mistranslation can be toxic to bacteria and mammalian cells, and can lead to heritable mutations. The great challenge for nature appears to be serine-for-alanine mistranslation, where even small amounts of this mistranslation cause severe neuropathologies in the mouse. To minimize serine-for-alanine mistranslation, powerful selective pressures developed to prevent mistranslation through a special editing activity imbedded within alanyl-tRNA synthetases (AlaRSs). However, serine-for-alanine mistranslation is so challenging that a separate, genome-encoded fragment of the editing domain of AlaRS is distributed throughout the Tree of Life to redundantly prevent serine-to-alanine mistranslation. Detailed X-ray structural and functional analysis shed light on why serine-for-alanine mistranslation is a universal problem, and on the selective pressures that engendered the appearance of AlaXps at the base of the Tree of Life. PMID:21930589

  14. The expression of prokaryotic tRNA genes in frog oocytes.

    PubMed Central

    Bossi, L; Ciampi, M S

    1983-01-01

    A tRNA gene cluster in Salmonella typhimurium includes the genes for tRNAArg, tRNAHis, tRNA1Leu and tRNAPro. DNA clones were constructed with different portions of this tRNA gene cluster. These clones were microinjected into the nuclei of Xenopus laevis oocytes and assayed for expression. Two of the bacterial tRNA genes (tRNAArg and tRNAPro) are transcribed at high rates and the primary transcripts are processed into mature tRNAs. Transcription and processing are largely independent of whether the two genes are injected individually or as part of a tRNA gene cluster. A third tRNA gene (tRNA1Leu) is expressed less efficiently. Synthesis of this tRNA is totally abolished by a deletion removing 22 bp in the first half of the tRNA1Leu coding sequence. The expression of the fourth tRNA gene (tRNAHis) is very inefficient and dependent upon the gene organization within the injected DNA. No significant tRNA synthesis is detected upon injection of a clone containing only the tRNAHis gene. Evidence is presented suggesting that the impaired expression of the tRNAHis gene is not caused by inefficient transcription, but rather by defective processing of the primary transcript. The prokaryotic tRNAs synthesized in the oocytes show a modification pattern that is specific of eukaryotic tRNAs. Overall, our results are consistent with the hypothesis that the intragenic signals for eukaryotic tRNA gene transcription have appeared early in evolution for reasons other than gene expression. Images PMID:6304627

  15. Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device

    PubMed Central

    Smith, Andrew M.; Abu-Shumays, Robin; Akeson, Mark; Bernick, David L.

    2015-01-01

    Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified Escherichia coli tRNAfMet and tRNALys, we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provide the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device. PMID:26157798

  16. Crystallographic snapshots of eukaryotic dimethylallyltransferase acting on tRNA: Insight into tRNA recognition and reaction mechanism

    SciTech Connect

    Zhou, Chun; Huang, Raven H.

    2009-01-15

    Hypermodifications near the anticodon of tRNA are fundamental for the efficiency and fidelity of protein synthesis. Dimethylallyltransferase (DMATase) catalyzes transfer of a dimethylallyl moiety from dimethylallyl pyrophosphate to N6 of A37 in certain tRNAs. Here we present the crystal structures of Saccharomyces cerevisiae DMATase-tRNA{sup Cys} complex in four distinct forms, which provide snapshots of the RNA modification reaction catalyzed by DMATase. The structures reveal that the enzyme recognizes the tRNA substrate through indirect sequence readout. The targeted nucleotide A37 flips out from the anticodon loop of tRNA and flips into a channel in DMATase, where it meets its reaction partner di methylallyl pyrophosphate, which enters the channel from the opposite end. Structural changes accompanying the transfer reaction taking place in the crystal result in disengagement of DMATase-tRNA interaction near the reaction center. In addition, structural comparison of DMATase in the complex with unliganded bacterial DMATase provides a molecular basis of ordered substrate binding by DMATase.

  17. Functional expansion of human tRNA synthetases achieved by structural inventions.

    PubMed

    Guo, Min; Schimmel, Paul; Yang, Xiang-Lei

    2010-01-21

    Known as an essential component of the translational apparatus, the aminoacyl-tRNA synthetase family catalyzes the first step reaction in protein synthesis, that is, to specifically attach each amino acid to its cognate tRNA. While preserving this essential role, tRNA synthetases developed other roles during evolution. Human tRNA synthetases, in particular, have diverse functions in different pathways involving angiogenesis, inflammation and apoptosis. The functional diversity is further illustrated in the association with various diseases through genetic mutations that do not affect aminoacylation or protein synthesis. Here we review the accumulated knowledge on how human tRNA synthetases used structural inventions to achieve functional expansions.

  18. CLP1 as a novel player in linking tRNA splicing to neurodegenerative disorders.

    PubMed

    Weitzer, Stefan; Hanada, Toshikatsu; Penninger, Josef M; Martinez, Javier

    2015-01-01

    Defects in RNA metabolic pathways are well-established causes for neurodegenerative disorders. Several mutations in genes involved in pre-messenger RNA (pre-mRNA) and tRNA metabolism, RNA stability and protein translation have been linked to motor neuron diseases. Our study on a mouse carrying a catalytically inactive version of the RNA kinase CLP1, a component of the tRNA splicing endonuclease complex, revealed a neurological disorder characterized by progressive loss of lower spinal motor neurons. Surprisingly, mutant mice accumulate a novel class of tRNA-derived fragments. In addition, patients with homozygous missense mutations in CLP1 (R140H) were recently identified who suffer from severe motor-sensory defects, cortical dysgenesis and microcephaly, and exhibit alterations in transfer RNA (tRNA) splicing. Here, we review functions of CLP1 in different RNA pathways and provide hypotheses on the role of the tRNA splicing machinery in the generation of tRNA fragments and the molecular links to neurodegenerative disorders. We further immerse the biology of tRNA splicing into topics of (t)RNA metabolism and oxidative stress, putting forward the idea that defects in tRNA processing leading to tRNA fragment accumulation might trigger the development of neurodegenerative diseases.

  19. Interaction of tRNA with MEK2 in pancreatic cancer cells

    PubMed Central

    Wang, Xiaoyun; Chow, Christina R.; Ebine, Kazumi; Lee, Jiyoung; Rosner, Marsha R.; Pan, Tao; Munshi, Hidayatullah G.

    2016-01-01

    Although the translational function of tRNA has long been established, extra translational functions of tRNA are still being discovered. We previously developed a computational method to systematically predict new tRNA-protein complexes and experimentally validated six candidate proteins, including the mitogen-activated protein kinase kinase 2 (MEK2), that interact with tRNA in HEK293T cells. However, consequences of the interaction between tRNA and these proteins remain to be elucidated. Here we tested the consequence of the interaction between tRNA and MEK2 in pancreatic cancer cell lines. We also generated disease and drug resistance-derived MEK2 mutants (Q60P, P128Q, S154F, E207K) to evaluate the function of the tRNA-MEK2 interaction. Our results demonstrate that tRNA interacts with the wild-type and mutant MEK2 in pancreatic cancer cells; furthermore, the MEK2 inhibitor U0126 significantly reduces the tRNA-MEK2 interaction. In addition, tRNA affects the catalytic activity of the wild type and mutant MEK2 proteins in different ways. Overall, our findings demonstrate the interaction of tRNA with MEK2 in pancreatic cancer cells and suggest that tRNA may impact MEK2 activity in cancer cells. PMID:27301426

  20. Epistatic selection of a sequence 5{prime} of the gene responsible for cystic fibrosis may account for the high frequency of this disease in the Caucasian population

    SciTech Connect

    Macek, M. Jr. |; Nash, E.; Cutting, G.R.

    1994-09-01

    Cystic fibrosis (CF) is one of the more common lethal autosomal recessive disorders in Caucasian populations. Numerous hypotheses including genetic drift, founder effect, sex ratio, segregation distortions and various forms of heterozygote advantage have been proposed to explain the relatively high frequency of CF alleles. The observation of high linkage disequilibrium between markers at the 5{prime} end of CFTR and mutations that cause CF raised the possibility of epistatic selection. CF-linked marker allele frequencies were determined in 417 elderly individuals from a stable Czech population that survived high levels of infant and childhood mortality in the pre-antibiotic era. These data were compared with allele frequencies of 646 contemporary newborns and 345 young adults drawn from the same population who had significantly lower mortality rates in the antibiotic era. Allele frequencies of markers CS7/Hhal and KM19/Pstl from the D7S23 locus are significantly different (p<0.05) between elderly female and male subjects in this population. Furthermore, there is a significant difference in the allele frequencies of marker CS7/Hhal when newborn females and elderly women are compared (p<0.05). Taken together, these data suggest that the allele status at the CS7 region influenced female survival in the period of high infant and childhood mortality in the pre-antibiotic era. Under this selective pressure, CFTR mutations that occurred on the {open_quotes}favorable{close_quotes} background would marginally increase in frequency in each successive generation and more ancient mutations residing on this background would become the most frequent in the general population.

  1. Stereochemical control over Mn(II)-Thio versus Mn(II)-Oxy coordination in adenosine 5 prime -O-(1-thiodiphosphate) complexes at the active site of creatine kinase

    SciTech Connect

    Smithers, G.W.; Sammons, R.D.; Goodhart, P.J.; LoBrutto, R.; Reed, G.H. )

    1989-02-21

    The stereochemical configurations of the Mn(II) complexes with the resolved epimers of adenosine 5{prime}-O-(1-thiodiphosphate) (ADP{alpha}S), bound at the active site of creatine kinase, have been determined in order to assess the relative strengths of enzymic stereoselectivity versus Lewis acid/base preferences in metal-ligand binding. Electron paramagnetic resonance (EPR) data have been obtained for Mn(II) in anion-stabilized, dead-end (transition-state analogue) complexes, in ternary enzyme-Mn{sup II}ADP{alpha}S complexes, and in the central complexes of the equilibrium mixture. The modes of coordination of Mn(II) at P{sub alpha} in the nitrate-stabilized, dead-end complexes with each epimer of ADP{alpha}S were ascertained by EPR measurements with (R{sub p})-({alpha}-{sup 17}O)ADP{alpha}S and (S{sub p})-({alpha}-{sup 17}O)ADP{alpha}S. A reduction in the magnitude of the {sup 55}Mn hyperfine coupling constant in the spectrum for the complex containing (S{sub p})-ADP{alpha}S is indicative of Mn(II)-thio coordination at P{sub alpha}. The results indicate that a strict discrimination for a unique configuration of the metal-nucleotide substrate is expressed upon binding of all of the substrates to form the active complex (or an analogue thereof). This enzymic stereoselectivity provides sufficient binding energy to overcome an intrinsic preference for the hard Lewis acid Mn(II) to coordinate to the hard Lewis base oxygen.

  2. Tyrosinase-positive oculocutaneous albinism in Southern African blacks: P gene-associated haplotypes suggest a major mutation in the 5{prime} region of the gene

    SciTech Connect

    Ramsay, M.; Stevens, G.; Beukering, J. van

    1994-09-01

    Tyrosinase-positive oculocutaneous albinism (ty-pos OCA) occurs with a prevalence of 1 in 3900 among Southern African (SA) blacks. The major contributors to morbidity and mortality are skin cancer and decreased visual acuity. Two distinct phenotypes occur, namely individuals with ephelides (darkly pigmented patches) and those without. There is complete concordance with regard to ephelus status among siblings. The disorder is linked to markers on chromosome 15q11.2-q12, and no obligatory cross-overs were observed with polymophic markers at the human homolog, P, of the mouse pink eyed dilute gene, p. Contrary to what has been shown for Caucasoid ty-pos OCA, this condition shows locus homogeneity among SA blacks. The P gene is an excellent candidate for ty-pos OCA and mutations in this gene will confirm its role in causing the common form of albinism in SA. Numerous P gene mutations have been described in other populations. In an attempt to detect mutations, the P gene cDNA was used to search for structural rearrangements or polymorphisms. Six polymorphisms (plR10/Scal, 912/Xbal, 912/HincII, 912/TaqI, 1412/TaqI [two systems] and 1412/HindIII) were detected with subclones of the P cDNA and haplotypes were determined in each family. None were clearly associated with an albinism-related rearrangement. However, strong linkage disequilibrium was observed with alleles at loci toward the 5{prime} region of the gene ({triangle}=0.65, 0.57 and 0.80 for the three polymorphisms detected with the 912 subclone), suggesting a major ty-pos OCA mutation in this region. Haplotype analysis provides evidence for a major mutation associated with the same haplotype in individuals with ephelides (8/12 OCA chromosomes) and those without ephelides (24:30). The presence of other ty-pos OCA associated haplotypes indicates several other less common mutations.

  3. Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity.

    PubMed

    Ramírez, Vicente; Gonzalez, Beatriz; López, Ana; Castelló, María José; Gil, María José; Etherington, Graham J; Zheng, Bo; Chen, Peng; Vera, Pablo

    2015-10-01

    tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA) signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response.

  4. Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity

    PubMed Central

    López, Ana; Castelló, María José; Gil, María José; Zheng, Bo; Chen, Peng; Vera, Pablo

    2015-01-01

    tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA) signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response. PMID:26492405

  5. A Rationally Engineered Misacylating Aminoacyl-Trna Synthetase

    SciTech Connect

    Bullock, T.L.; Rodriguez-Hernandez, A.; Corigliano, E.M.; Perona, J.J.

    2009-05-12

    Information transfer from nucleic acid to protein is mediated by aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K{sub M} of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA{sup Gln} by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA{sup Gln} by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS-tRNA{sup Gln} complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid-RNA pairing.

  6. An alanine tRNA gene cluster from Nephila clavipes.

    PubMed

    Luciano, E; Candelas, G C

    1996-06-01

    We report the sequence of a 2.3-kb genomic DNA fragment from the orb-web spider, Nephila clavipes (Nc). The fragment contains four regions of high homology to tRNA(Ala). The members of this irregularly spaced cluster of genes are oriented in the same direction and have the same anticodon (GCA), but their sequence differs at several positions. Initiation and termination signals, as well as consensus intragenic promoter sequences characteristic of tRNA genes, have been identified in all genes. tRNA(Ala) are involved in the regulation of the fibroin synthesis in the large ampullate Nc glands.

  7. Every little piece counts: the many faces of tRNA transcripts.

    PubMed

    Lalaouna, David; Carrier, Marie-Claude; Massé, Eric

    2015-01-01

    For over half a century, tRNAs have been exclusively known as decoders of genomic information. However, recent reports evidenced that tRNA transcripts are also bearers of functional RNAs, which are able to execute various tasks through an array of mechanisms. Here, we succinctly review the diversity and functions of RNAs deriving from tRNA loci. PMID:26595434

  8. Towards a comprehensive picture of alloacceptor tRNA remolding in metazoan mitochondrial genomes

    PubMed Central

    Sahyoun, Abdullah H.; Hölzer, Martin; Jühling, Frank; Höner zu Siederdissen, Christian; Al-Arab, Marwa; Tout, Kifah; Marz, Manja; Middendorf, Martin; Stadler, Peter F.; Bernt, Matthias

    2015-01-01

    Remolding of tRNAs is a well-documented process in mitochondrial genomes that changes the identity of a tRNA. It involves a duplication of a tRNA gene, a mutation that changes the anticodon and the loss of the ancestral tRNA gene. The net effect is a functional tRNA that is more closely related to tRNAs of a different alloacceptor family than to tRNAs with the same anticodon in related species. Beyond being of interest for understanding mitochondrial tRNA function and evolution, tRNA remolding events can lead to artifacts in the annotation of mitogenomes and thus in studies of mitogenomic evolution. Therefore, it is important to identify and catalog these events. Here we describe novel methods to detect tRNA remolding in large-scale data sets and apply them to survey tRNA remolding throughout animal evolution. We identify several novel remolding events in addition to the ones previously mentioned in the literature. A detailed analysis of these remoldings showed that many of them are derived from ancestral events. PMID:26227972

  9. Quality Control Pathways for Nucleus-Encoded Eukaryotic tRNA Biosynthesis and Subcellular Trafficking

    PubMed Central

    Huang, Hsiao-Yun

    2015-01-01

    tRNAs perform an essential role in translating the genetic code. They are long-lived RNAs that are generated via numerous posttranscriptional steps. Eukaryotic cells have evolved numerous layers of quality control mechanisms to ensure that the tRNAs are appropriately structured, processed, and modified. We describe the known tRNA quality control processes that check tRNAs and correct or destroy aberrant tRNAs. These mechanisms employ two types of exonucleases, CCA end addition, tRNA nuclear aminoacylation, and tRNA subcellular traffic. We arrange these processes in order of the steps that occur from generation of precursor tRNAs by RNA polymerase (Pol) III transcription to end maturation and modification in the nucleus to splicing and additional modifications in the cytoplasm. Finally, we discuss the tRNA retrograde pathway, which allows tRNA reimport into the nucleus for degradation or repair. PMID:25848089

  10. Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics.

    PubMed

    Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K

    2015-12-15

    Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear-cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. PMID:26680305

  11. Crystal structure of tRNA m1G9 methyltransferase Trm10: insight into the catalytic mechanism and recognition of tRNA substrate

    PubMed Central

    Shao, Zhenhua; Yan, Wei; Peng, Junhui; Zuo, Xiaobing; Zou, Yang; Li, Fudong; Gong, Deshun; Ma, Rongsheng; Wu, Jihui; Shi, Yunyu; Zhang, Zhiyong; Teng, Maikun; Li, Xu; Gong, Qingguo

    2014-01-01

    Transfer RNA (tRNA) methylation is necessary for the proper biological function of tRNA. The N1 methylation of guanine at Position 9 (m1G9) of tRNA, which is widely identified in eukaryotes and archaea, was found to be catalyzed by the Trm10 family of methyltransferases (MTases). Here, we report the first crystal structures of the tRNA MTase spTrm10 from Schizosaccharomyces pombe in the presence and absence of its methyl donor product S-adenosyl-homocysteine (SAH) and its ortholog scTrm10 from Saccharomyces cerevisiae in complex with SAH. Our crystal structures indicated that the MTase domain (the catalytic domain) of the Trm10 family displays a typical SpoU-TrmD (SPOUT) fold. Furthermore, small angle X-ray scattering analysis reveals that Trm10 behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. We also performed tRNA MTase assays and isothermal titration calorimetry experiments to investigate the catalytic mechanism of Trm10 in vitro. In combination with mutational analysis and electrophoretic mobility shift assays, our results provide insights into the substrate tRNA recognition mechanism of Trm10 family MTases. PMID:24081582

  12. tRNA evolution from the proto-tRNA minihelix world

    PubMed Central

    Root-Bernstein, Robert; Kim, Yunsoo; Sanjay, Adithya; Burton, Zachary F.

    2016-01-01

    ABSTRACT Multiple models have been advanced for the evolution of cloverleaf tRNA. Here, the conserved archaeal tRNA core (75-nt) is posited to have evolved from ligation of three proto-tRNA minihelices (31-nt) and two-symmetrical 9-nt deletions within joined acceptor stems (93 – 18 = 75-nt). The primary evidence for this conclusion is that the 5-nt stem 7-nt anticodon loop and the 5-nt stem 7-nt T loop are structurally homologous and related by coding sequence. We posit that the D loop was generated from a third minihelix (31-nt) in which the stem and loop became rearranged after 9-nt acceptor stem deletions and cloverleaf folding. The most 3´-5-nt segment of the D loop and the 5-nt V loop are apparent remnants of the joined acceptor stems (14 – 9 = 5-nt). Before refolding in the tRNA cloverleaf, we posit that the 3′-5-nt segment of the D loop and the 5-nt V loop were paired, and, in the tRNA cloverleaf, frequent pairing of positions 29 (D loop) and 47 (V loop) remains (numbered on a 75-nt tRNA cloverleaf core). Amazingly, after >3.5 billion years of evolutionary pressure on the tRNA cloverleaf structure, a model can be constructed that convincingly describes the genesis of 75/75-nt conserved archaeal tRNA core positions. Judging from the tRNA structure, cloverleaf tRNA appears to represent at least a second-generation scheme (and possibly a third-generation scheme) that replaced a robust 31-nt minihelix protein-coding system, evidence for which is preserved in the cloverleaf structure. Understanding tRNA evolution provides insights into ribosome and rRNA evolution. PMID:27636862

  13. Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases

    PubMed Central

    Byrne, Robert T.; Jenkins, Huw T.; Peters, Daniel T.; Whelan, Fiona; Stowell, James; Aziz, Naveed; Kasatsky, Pavel; Rodnina, Marina V.; Koonin, Eugene V.; Konevega, Andrey L.; Antson, Alfred A.

    2015-01-01

    The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNAPhe and tRNATrp show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160° rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids (“binding signatures”) together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal “recognition” domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold. PMID:25902496

  14. Involvement of imported tRNA in intramitochondrial translation. [Tetrahymena

    SciTech Connect

    Suyama, Y.

    1981-01-01

    These studies show that only 10 out of 36 mitochondrial tRNAs hybridize to mtDNA. Consistent with previous observations, Arg, Ile, Lys, Val tRNAs must be imported cytoplasmic tRNAs, since these tRNAs do not hybridize to mtDNA. The evident indicates that these imported tRNAs in Tetrahymena mitochondria are not contaminating cytoplasmic tRNAs in our mitochondrial preparations. The conclusion that they function in intramitochondrial translation is based on the demonstration that all the native and imported tRNAs are associated with the functinal mitochondrial 80S monosome as well as with carefully washed 55S subunits. As expected if they function in translation, all these tRNAs on the ribosomes should become acylated when mitochondria are engaged in protein synthesis. From the codon recognition patterns determined previously, it is quite probable that Tetrahymena mitochondrial translation system differs from mammalian and fungal mitochondrial systems. The mechanisms for transporting tRNA into mitochondria is not known. However, it was proposed earlier that the corresponding tRNA synthetase may act as transport protein.

  15. A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

    SciTech Connect

    Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi )

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

  16. The selenocysteine-inserting opal suppressor serine tRNA from E. coli is highly unusual in structure and modification.

    PubMed Central

    Schön, A; Böck, A; Ott, G; Sprinzl, M; Söll, D

    1989-01-01

    Selenocysteine is cotranslationally incorporated into selenoproteins in a unique pathway involving tRNA mediated suppression of a UGA nonsense codon (1-3). The DNA sequence of the gene for this suppressor tRNA from Escherichia coli predicts unusual features of the gene product (4). We determined the sequence of this serine tRNA (tRNA(UCASer]. It is the longest tRNA (95 nt) known to date with an acceptor stem of 8 base pairs and lacks some of the 'invariant' nucleotides found in other tRNAs. It is the first E. coli tRNA that contains the hypermodified nucleotide i6A, adjacent to the UGA-recognizing anticodon UCA. The implications of the unusual structure and modification of this tRNA on recognition by seryl-tRNA synthetase, by tRNA modifying enzymes, and on codon recognition are discussed. PMID:2529478

  17. Examining the Gm18 and m1G Modification Positions in tRNA Sequences

    PubMed Central

    Subramanian, Mayavan; Srinivasan, Thangavelu

    2014-01-01

    The tRNA structure contains conserved modifications that are responsible for its stability and are involved in the initiation and accuracy of the translation process. tRNA modification enzymes are prevalent in bacteria, archaea, and eukaryotes. tRNA Gm18 methyltransferase (TrmH) and tRNA m1G37 methyltransferase (TrmD) are prevalent and essential enzymes in bacterial populations. TrmH involves itself in methylation process at the 2'-OH group of ribose at the 18th position of guanosine (G) in tRNAs. TrmD methylates the G residue next to the anticodon in selected tRNA subsets. Initially, m1G37 modification was reported to take place on three conserved tRNA subsets (tRNAArg, tRNALeu, tRNAPro); later on, few archaea and eukaryotes organisms revealed that other tRNAs also have the m1G37 modification. The present study reveals Gm18, m1G37 modification, and positions of m1G that take place next to the anticodon in tRNA sequences. We selected extremophile organisms and attempted to retrieve the m1G and Gm18 modification bases in tRNA sequences. Results showed that the Gm18 modification G residue occurs in all tRNA subsets except three tRNAs (tRNAMet, tRNAPro, tRNAVal). Whereas the m1G37 modification base G is formed only on tRNAArg, tRNALeu, tRNAPro, and tRNAHis, the rest of the tRNAs contain adenine (A) next to the anticodon. Thus, we hypothesize that Gm18 modification and m1G modification occur irrespective of a G residue in tRNAs. PMID:25031570

  18. The high content of natural suppressor serine tRNA in dystrophic mouse muscle.

    PubMed Central

    Hitaka, T; Mizutani, T; Watanabe, K; Totsuka, T

    1990-01-01

    In order to gain an insight into the pathogenesis of mouse muscular dystrophy, we investigated the natural suppressor serine tRNA. The natural suppressor seryl-tRNA was distinguished from the other seryl-tRNAs on the basis of its specific property of being converted into phosphoseryl-tRNA by a tRNA kinase. On a wet-weight basis, the content of total tRNA in dystrophic muscles was 47% of that in normal muscles. Although the serine-accepting activities of tRNA were similar in muscles of 3-month-old dystrophic and normal mice, the ratio of [32P]phosphoseryl-tRNA (suppressor tRNA) to the total serine tRNA was significantly enhanced in dystrophic muscles compared with that in normal muscles. This high content of suppressor tRNA in dystrophic muscles was further confirmed by dot-blot hybridization experiments with the DNA probes CGTAGTCGGCAGGAT and CGCCCGAAAGGTGGAA for major tRNA(IGASer) and suppressor tRNA respectively. At the early postnatal age of 3 weeks, when only a week had elapsed since the first manifestation of the dystrophic symptom (hindleg dragging), the ratio of suppressor tRNA to major tRNAs in dystrophic hindleg muscles was abnormally increased. Thereafter it decreased with age in normal mice but remained almost unchanged in dystrophic mice. Consequently, at 3 months old, it was 1.7 times higher in dystrophic than in normal mice. The suppressor tRNA is now accepted to play a role in the synthesis of glutathione peroxidase. The present study showed that the content of this enzyme was abnormally elevated in dystrophic mice. Previously we had demonstrated that the docosahexaenoic (C22:6) acid content in phospholipids was decreased, possibly resulting from the enhanced oxidative milieu caused by the dystrophic condition. Thus far, the findings suggest that an increase in the contents of suppressor tRNA and glutathione peroxidase in dystrophic muscle may have been secondarily induced by such a highly oxidative state in the dystrophic condition. However, it is

  19. Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding

    PubMed Central

    Ozanick, Sarah G.; Bujnicki, Janusz M.; Sem, Daniel S.; Anderson, James T.

    2007-01-01

    In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNAiMet. The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-l-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNAiMet, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present. PMID:17932071

  20. Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements

    PubMed Central

    Jühling, Frank; Pütz, Joern; Bernt, Matthias; Donath, Alexander; Middendorf, Martin; Florentz, Catherine; Stadler, Peter F.

    2012-01-01

    Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit ‘bizarre’ secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading ‘pseudogenes’, even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders. PMID:22139921

  1. Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation.

    PubMed

    Hussain, Tanweer; Llácer, Jose L; Wimberly, Brian T; Kieft, Jeffrey S; Ramakrishnan, V

    2016-09-22

    In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition. PMID:27662086

  2. The T box mechanism: tRNA as a regulatory molecule

    PubMed Central

    Green, Nicholas J.; Grundy, Frank J.; Henkin, Tina M.

    2009-01-01

    The T box mechanism is widely used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA synthetase genes and genes involved in amino acid biosynthesis and uptake. Binding of a specific uncharged tRNA to a riboswitch element in the nascent transcript causes a structural change in the transcript that promotes expression of the downstream coding sequence. In most cases, this occurs by stabilization of an antiterminator element that competes with formation of a terminator helix. Specific tRNA recognition by the nascent transcript results in increased expression of genes important for tRNA aminoacylation in response to decreased pools of charged tRNA. PMID:19932103

  3. Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding

    SciTech Connect

    Dunkle, Jack A.; Wang, Leyi; Feldman, Michael B.; Pulk, Arto; Chen, Vincent B.; Kapral, Gary J.; Noeske, Jonas; Richardson, Jane S.; Blanchard, Scott C.; Cate, Jamie H. Doudna

    2011-09-06

    During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of {approx}3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

  4. Extensive and evolutionarily persistent mitochondrial tRNA editing in Velvet Worms (phylum Onychophora).

    PubMed

    Segovia, Romulo; Pett, Walker; Trewick, Steve; Lavrov, Dennis V

    2011-10-01

    Mitochondrial genomes of onychophorans (velvet worms) present an interesting problem: Some previous studies reported them lacking several transfer RNA (tRNA) genes, whereas others found that all their tRNA genes were present but severely reduced. To resolve this discrepancy, we determined complete mitochondrial DNA (mtDNA) sequences of the onychophorans Oroperipatus sp. and Peripatoides sympatrica as well as cDNA sequences from 14 and 10 of their tRNAs, respectively. We show that tRNA genes in these genomes are indeed highly reduced and encode truncated molecules, which are restored to more conventional structures by extensive tRNA editing. During this editing process, up to 34 nucleotides are added to the tRNA sequences encoded in Oroperipatus sp. mtDNA, rebuilding the aminoacyl acceptor stem, the TΨC arm, and in some extreme cases, the variable arm and even a part of the anticodon stem. The editing is less extreme in P. sympatrica in which at least a part of the TΨC arm is always encoded in mtDNA. When the entire TΨC arm is added de novo in Oroperipatus sp., the sequence of this arm is either identical or similar among different tRNA species, yet the sequences show substantial variation for each tRNA. These observations suggest that the arm is rebuilt, at least in part, by a template-independent mechanism and argue against the alternative possibility that tRNA genes or their parts are imported from the nucleus. By contrast, the 3' end of the aminoacyl acceptor stem is likely restored by a template-dependent mechanism. The extreme tRNA editing reported here has been preserved for >140 My as it was found in both extant families of onychophorans. Furthermore, a similar type of tRNA editing may be present in several other groups of arthropods, which show a high degree of tRNA gene reduction in their mtDNA. PMID:21546355

  5. Extensive and evolutionarily persistent mitochondrial tRNA editing in Velvet Worms (phylum Onychophora).

    PubMed

    Segovia, Romulo; Pett, Walker; Trewick, Steve; Lavrov, Dennis V

    2011-10-01

    Mitochondrial genomes of onychophorans (velvet worms) present an interesting problem: Some previous studies reported them lacking several transfer RNA (tRNA) genes, whereas others found that all their tRNA genes were present but severely reduced. To resolve this discrepancy, we determined complete mitochondrial DNA (mtDNA) sequences of the onychophorans Oroperipatus sp. and Peripatoides sympatrica as well as cDNA sequences from 14 and 10 of their tRNAs, respectively. We show that tRNA genes in these genomes are indeed highly reduced and encode truncated molecules, which are restored to more conventional structures by extensive tRNA editing. During this editing process, up to 34 nucleotides are added to the tRNA sequences encoded in Oroperipatus sp. mtDNA, rebuilding the aminoacyl acceptor stem, the TΨC arm, and in some extreme cases, the variable arm and even a part of the anticodon stem. The editing is less extreme in P. sympatrica in which at least a part of the TΨC arm is always encoded in mtDNA. When the entire TΨC arm is added de novo in Oroperipatus sp., the sequence of this arm is either identical or similar among different tRNA species, yet the sequences show substantial variation for each tRNA. These observations suggest that the arm is rebuilt, at least in part, by a template-independent mechanism and argue against the alternative possibility that tRNA genes or their parts are imported from the nucleus. By contrast, the 3' end of the aminoacyl acceptor stem is likely restored by a template-dependent mechanism. The extreme tRNA editing reported here has been preserved for >140 My as it was found in both extant families of onychophorans. Furthermore, a similar type of tRNA editing may be present in several other groups of arthropods, which show a high degree of tRNA gene reduction in their mtDNA.

  6. G to A substitution in 5{prime} donor splice site of introns 18 and 48 of COL1A1 gene of type I collagen results in different splicing alternatives in osteogenesis imperfecta type I cell strains

    SciTech Connect

    Willing, M.; Deschenes, S.

    1994-09-01

    We have identified a G to A substitution in the 5{prime} donor splice site of intron 18 of one COL1A1 allele in two unrelated families with osteogenesis imperfecta (OI) type I. A third OI type I family has a G to A substitution at the identical position in intron 48 of one COL1A1 allele. Both mutations abolish normal splicing and lead to reduced steady-state levels of mRNA from the mutant COL1A1 allele. The intron 18 mutation leads to both exon 18 skipping in the mRNA and to utilization of a single alternative splice site near the 3{prime} end of exon 18. The latter results in deletion of the last 8 nucleotides of exon 18 from the mRNA, a shift in the translational reading-frame, and the creation of a premature termination codon in exon 19. Of the potential alternative 5{prime} splice sites in exon 18 and intron 18, the one utilized has a surrounding nucleotide sequence which most closely resembles that of the natural splice site. Although a G to A mutation was detected at the identical position in intron 48 of one COL1A1 allele in another OI type I family, nine complex alternative splicing patterns were identified by sequence analysis of cDNA clones derived from fibroblast mRNA from this cell strain. All result in partial or complete skipping of exon 48, with in-frame deletions of portions of exons 47 and/or 49. The different patterns of RNA splicing were not explained by their sequence homology with naturally occuring 5{prime} splice sites, but rather by recombination between highly homologous exon sequences, suggesting that we may not have identified the major splicing alternative(s) in this cell strain. Both G to A mutations result in decreased production of type I collagen, the common biochemical correlate of OI type I.

  7. Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives.

    PubMed

    Graifer, D M; Babkina, G T; Matasova, N B; Vladimirov, S N; Karpova, G G; Vlassov, V V

    1989-07-01

    A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

  8. tRNA thiolation links translation to stress responses in Saccharomyces cerevisiae.

    PubMed

    Damon, Jadyn R; Pincus, David; Ploegh, Hidde L

    2015-01-15

    Although tRNA modifications have been well catalogued, the precise functions of many modifications and their roles in mediating gene expression are still being elucidated. Whereas tRNA modifications were long assumed to be constitutive, it is now apparent that the modification status of tRNAs changes in response to different environmental conditions. The URM1 pathway is required for thiolation of the cytoplasmic tRNAs tGlu(UUC), tGln(UUG), and tLys(UUU) in Saccharomyces cerevisiae. We demonstrate that URM1 pathway mutants have impaired translation, which results in increased basal activation of the Hsf1-mediated heat shock response; we also find that tRNA thiolation levels in wild-type cells decrease when cells are grown at elevated temperature. We show that defects in tRNA thiolation can be conditionally advantageous, conferring resistance to endoplasmic reticulum stress. URM1 pathway proteins are unstable and hence are more sensitive to changes in the translational capacity of cells, which is decreased in cells experiencing stresses. We propose a model in which a stress-induced decrease in translation results in decreased levels of URM1 pathway components, which results in decreased tRNA thiolation levels, which further serves to decrease translation. This mechanism ensures that tRNA thiolation and translation are tightly coupled and coregulated according to need. PMID:25392298

  9. A subset of conserved tRNA genes in plastid DNA of nongreen plants.

    PubMed Central

    Lohan, A J; Wolfe, K H

    1998-01-01

    The plastid genome of the nonphotosynthetic parasitic plant Epifagus virginiana contains only 17 of the 30 tRNA genes normally found in angiosperm plastid DNA. Although this is insufficient for translation, the genome is functional, so import of cytosolic tRNAs into plastids has been suggested. This raises the question of whether the tRNA genes that remain in E. virginiana plastid DNA are active or have just fortuitously escaped deletion. We report the sequences of 20 plastid tRNA loci from Orobanche minor, which shares a nonphotosynthetic ancestor with E. virginiana. The two species have 9 intact tRNA genes in common, the others being defunct in one or both species. The intron-containing trnLUAA gene is absent from E. virginiana, but it is intact, transcribed, and spliced in O. minor. The shared intact genes are better conserved than intergenic sequences, which indicates that these genes are being maintained by natural selection and, therefore, must be functional. For the most part, the tRNA species conserved in nonphotosynthetic plastids are also those that have never been found to be imported in plant mitochondria, which suggests that the same rules may govern tRNA import in the two organelles. A small photosynthesis gene, psbI, is still intact in O. minor, and computer simulations show that some small nonessential genes have an appreciable chance of escaping deletion. PMID:9725858

  10. Exploring GpG bases next to anticodon in tRNA subsets

    PubMed Central

    Srinivasan, Thangavelu; Kumaran, Kubendiran; Selvakumar, Rajendran; Velmurugan, Devadasan; Sudarsanam, Dorairaj

    2013-01-01

    Transfer RNA (tRNA) structure, modifications and functions are evolutionary and established in bacteria, archaea and eukaryotes. Typically the tRNA modifications are indispensable for its stability and are required for decoding the mRNA into amino acids for protein synthesis. A conserved methylation has been located on the anticodon loop specifically at the 37th position and it is next to the anticodon bases. This modification is called as m1G37 and it is catalyzed by tRNA (m1G37) methyltransferase (TrmD). It is deciphered that G37 positions occur on few additional amino acids specific tRNA subsets in bacteria. Furthermore, Archaea and Eukaryotes have more number of tRNA subsets which contains G37 position next to the anticodon and the G residue are located at different positions such as G36, G37, G38, 39, and G40. In eight bacterial species, G (guanosine) residues are presents at the 37th and 38th position except three tRNA subsets having G residues at 36th and 39th positions. Therefore we propose that m1G37 modification may be feasible at 36th, 37th, 38th, 39th and 40th positions next to the anticodon of tRNAs. Collectively, methylation at G residues close to the anticodon may be possible at different positions and without restriction of anticodon 3rd base A, C, U or G. PMID:23847401

  11. Impaired tRNA nuclear export links DNA damage and cell-cycle checkpoint.

    PubMed

    Ghavidel, Ata; Kislinger, Thomas; Pogoutse, Oxana; Sopko, Richelle; Jurisica, Igor; Emili, Andrew

    2007-11-30

    In response to genotoxic stress, cells evoke a plethora of physiological responses collectively aimed at enhancing viability and maintaining the integrity of the genome. Here, we report that unspliced tRNA rapidly accumulates in the nuclei of yeast Saccharomyces cerevisiae after DNA damage. This response requires an intact MEC1- and RAD53-dependent signaling pathway that impedes the nuclear export of intron-containing tRNA via differential relocalization of the karyopherin Los1 to the cytoplasm. The accumulation of unspliced tRNA in the nucleus signals the activation of Gcn4 transcription factor, which, in turn, contributes to cell-cycle arrest in G1 in part by delaying accumulation of the cyclin Cln2. The regulated nucleocytoplasmic tRNA trafficking thus constitutes an integral physiological adaptation to DNA damage. These data further illustrate how signal-mediated crosstalk between distinct functional modules, namely, tRNA nucleocytoplasmic trafficking, protein synthesis, and checkpoint execution, allows for functional coupling of tRNA biogenesis and cell-cycle progression.

  12. TFAM 1.0: an online tRNA function classifier.

    PubMed

    Tåquist, Helena; Cui, Yuanyuan; Ardell, David H

    2007-07-01

    We have earlier published an automated statistical classifier of tRNA function called TFAM. Unlike tRNA gene-finders, TFAM uses information from the total sequences of tRNAs and not just their anticodons to predict their function. Therefore TFAM has an advantage in predicting initiator tRNAs, the amino acid charging identity of nonstandard tRNAs such as suppressors, and the former identity of pseudo-tRNAs. In addition, TFAM predictions are robust to sequencing errors and useful for the statistical analysis of tRNA sequence, function and evolution. Earlier versions of TFAM required a complicated installation and running procedure, and only bacterial tRNA identity models were provided. Here we describe a new version of TFAM with both a Web Server interface and simplified standalone installation. New TFAM models are available including a proteobacterial model for the bacterial lysylated isoleucine tRNAs, making it now possible for TFAM to correctly classify all tRNA genes for some bacterial taxa. First-draft eukaryotic and archaeal models are also provided making initiator tRNA prediction easily accessible genes to any researcher or genome sequencing effort. The TFAM Web Server is available at http://tfam.lcb.uu.se.

  13. A novel label-free optical cysteine sensor based on the competitive oxidation reaction catalyzed by G-quadruplex halves.

    PubMed

    Su, Haichao; Qiao, Fengmin; Duan, Ruihuan; Chen, Lijian; Ai, Shiyun

    2013-05-15

    A sensitive and selective colorimetric detection method for Cysteine (Cys) was established in this paper. The detection mechanism is based on the oxidation of Cys by H2O2, which prevents the catalysis of the 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)-H2O2 reaction by G-quadruplex halves. With the addition of Cys, the amount of the blue-green-colored free-radical cation (ABTS(·+)) was reduced. The absorbance of ABTS(+) at 421nm weakened as the color of the solution changed from blue-green to colorless. The concentration of Cys can be determined by monitoring this competitive reaction with the naked eye or using a UV-vis spectrometer. The calibration curve showed that the net absorption value at 421nm linearly increased over the Cys concentration range of 0.005-100μM with a detection limit of 5nM. Furthermore, amino acids other than Cys cannot mediate the color change under the identical conditions because of the absence of thiol groups, thereby suggesting the selectivity towards Cys of the proposed method. The optical sensor is high selective, which is important for the determination of Cys in serum samples. The assay shows great potential for its practical application as a disease-associated indicator which could satisfy the need for amino acid determination in fields such as food processing, biochemistry, pharmaceuticals, and clinical analysis. PMID:23333922

  14. Gene expression changes induced by Trypanosoma cruzi shed microvesicles in mammalian host cells: relevance of tRNA-derived halves.

    PubMed

    Garcia-Silva, Maria R; Cabrera-Cabrera, Florencia; das Neves, Roberta Ferreira Cura; Souto-Padrón, Thaís; de Souza, Wanderley; Cayota, Alfonso

    2014-01-01

    At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms including Trypanosoma cruzi which lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles from T. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation of T. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted by T. cruzi could be relevant players in early events of the T. cruzi host cell interplay.

  15. Systematic screening for mutations in the 5{prime}-regulatory region of the human dopamine D{sub 1} receptor (DRD1) gene in patients with schizophrenia and bipolar affective disorder

    SciTech Connect

    Cichon, S.; Noethen, M.M.; Stoeber, G.

    1996-07-26

    A possible dysregulation of dopaminergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study we systematically searched for the presence of mutations in the 5{prime}-flanking region of the dopamine D{sub 1} receptor (DRD1) gene. This region has previously been shown to contain a functional promoter. We investigated 119 unrelated individuals (including 36 schizophrenic patients, 38 bipolar affective patients, and 45 healthy controls) using single-strand conformation analysis (SSCA). Eleven overlapping PCR fragments covered 2,189 bp of DNA sequence. We identified six single base substitutions: -2218T/C, -2102C/A, -2030T/C, -1992G/A, -1251G/C, and -800T/C. None of the mutations was found to be located in regions which have important influence on the level of transcriptional activity. Allele frequencies were similar in patients and controls, indicating that genetic variation in the 5{prime}-regulatory region of the DRD1 gene is unlikely to play a frequent, major role in the genetic predisposition to either schizophrenia or bipolar affective disorder. 31 refs., 3 tabs.

  16. Structural analysis of the 5 prime flanking region of the. beta. -globin gene in African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa

    SciTech Connect

    Chebloune, Y.; Pagnier, J.; Trabuchet, G.; Faure, C.; Verdier, G.; Labie, D.; Nigon, V. )

    1988-06-01

    Haplotype analysis of the {beta}-globin gene cluster shows two regions of DNA characterized by nonrandom association of restriction site polymorphisms. These regions are separated by a variable segment containing the repeated sequences (ATTTT){sub n} and (AT){sub x}T{sub y}, which might be involved in recombinational events. Studies of haplotypes linked to the sickle cell gene in Africa provide strong argument for three origins of the mutation: Benin, Senegal, and the Central African Republic. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT){sub n} and (AT){sub x}T{sub y} repeats. The authors found three additional structures for (AT){sub x}T{sub y} correlating with the geographic origin of the patients. Ten other nucleotide positions, 5{prime} and 3{prime} to the (AT){sub x}T{sub y} copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5{prime} flanking region of the {beta}-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.

  17. Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes

    PubMed Central

    Mix, Heiko; Zhang, Yan; Saira, Kazima; Glass, Richard S; Berry, Marla J; Gladyshev, Vadim N; Hatfield, Dolph L

    2007-01-01

    Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA[Ser]Sec as substrates to generate selenocysteyl-tRNA[Ser]Sec. Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA[Ser]Sec, seryl-tRNA synthetase, O-phosphoseryl-tRNA[Ser]Sec kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA[Ser]Sec kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins. PMID:17194211

  18. The binding sites for tRNA on eukaryotic ribosomes.

    PubMed

    Leader, D P; Machray, G C

    1975-07-01

    We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.

  19. The binding sites for tRNA on eukaryotic ribosomes.

    PubMed Central

    Leader, D P; Machray, G C

    1975-01-01

    We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes. PMID:1098024

  20. The anticodon of the maize chloroplast gene for tRNA Leu UAA is split by a large intron.

    PubMed Central

    Steinmetz, A; Gubbins, E J; Bogorad, L

    1982-01-01

    The maize chloroplast gene encoding tRNA Leu UAA has been sequenced. It contains a 458 base pair intron between the first and second bases of the anticodon. The tRNA is 88 nucleotides long (the 3'-terminal CCA sequence included which, however, is not encoded by the gene) and differs in only four nucleotides (modified nucleotides are not considered) from the corresponding isoacceptor from bean chloroplasts. The unusual position of the intron in this maize chloroplast tRNA gene suggests a splicing model different from that generally accepted for eukaryotic split tRNA genes. Images PMID:6285285

  1. A yeast arginine specific tRNA is a remnant aspartate acceptor

    PubMed Central

    Fender, Aurélie; Geslain, Renaud; Eriani, Gilbert; Giegé, Richard; Sissler, Marie; Florentz, Catherine

    2004-01-01

    High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNAAsp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA4Arg. This tRNA has a sequence strikingly similar to that of tRNAAsp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA4Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA4Arg into an aspartate acceptor, as efficient as tRNAAsp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA4Arg. The latent aspartate acceptor capacity in a contemporary tRNAArg leads to the proposal of an evolutionary link between tRNA4Arg and tRNAAsp genes. PMID:15452274

  2. The nuclear tRNA aminoacylation-dependent pathway may be the principal route used to export tRNA from the nucleus in Saccharomyces cerevisiae.

    PubMed

    Steiner-Mosonyi, Marta; Mangroo, Dev

    2004-03-15

    Nuclear tRNA export in Saccharomyces cerevisiae has been proposed to involve three pathways, designated Los1p-dependent, Los1p-independent nuclear aminoacylation-dependent, and Los1p- and nuclear aminoacylation-independent. Here, a comprehensive biochemical analysis was performed to identify tRNAs exported by the aminoacylation-dependent and -independent pathways of S. cerevisiae. Interestingly, the major tRNA species of at least 19 families were found in the aminoacylated form in the nucleus. tRNAs known to be exported by the export receptor Los1p were also aminoacylated in the nucleus of both wild-type and mutant Los1p strains. FISH (fluorescence in situ hybridization) analyses showed that tRNA(Tyr) co-localizes with the U18 small nucleolar RNA in the nucleolus of a tyrosyl-tRNA synthetase mutant strain defective in nuclear tRNA(Tyr) export because of a block in nuclear tRNA(Tyr) aminoacylation. tRNA(Tyr) was also found in the nucleolus of a utp8 mutant strain defective in nuclear tRNA export but not nuclear tRNA aminoacylation. These results strongly suggest that the nuclear aminoacylation-dependent pathway is principally responsible for tRNA export in S. cerevisiae and that Los1p is an export receptor of this pathway. It is also likely that in mammalian cells tRNAs are mainly exported from the nucleus by the nuclear aminoacylation-dependent pathway. In addition, the data are consistent with the idea that nuclear aminoacylation is used as a quality control mechanism for ensuring nuclear export of only mature and functional tRNAs, and that this quality assurance step occurs in the nucleolus.

  3. Analysis of the UV photolesion spectrum in the glnU tRNA gene of Escherichia coli: A role in the generation of nonsense suppressor mutations by ultraviolet light

    SciTech Connect

    Garvey, N.

    1988-01-01

    UV-induced ochre suppressor mutations arise from a GC {yields} AT transition at the 5{prime} end of the anticodon-encoding sequence CAA in the glutamine tRNA gene glnU. This site is a conditional hotspot for UV mutagenesis. When post-irradiation medium prohibits protein synthesis, suppressor mutation yields are low, equivalent to that for other types of mutations. However, when incubation medium supports protein synthesis, suppressor mutations are ten times more frequent than others. This presumably reflects excision repair failure at this site under these conditions. The lesion remains in the DNA and targets a transition mutation by SOS processing during the subsequent round of replication. The specific and rapid loss of potential suppressor mutations when protein synthesis is inhibited is termed mutation frequency decline (MFD). In an effort to understand MFD, we subcloned the glnU gene onto a high copy number plasmid and measured the types and frequencies of UV photolesions at the anticodon-encoding trinucleotide and in thirty-three base pairs flanking it.

  4. Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues.

    PubMed

    Fung, Angela Wai Shan; Ebhardt, H Alexander; Krishnakumar, Kollappillil S; Moore, Jack; Xu, Zhizhong; Strazewski, Peter; Fahlman, Richard P

    2014-07-01

    Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.

  5. Strategies for investigating nuclear-cytoplasmic tRNA dynamics in yeast and mammalian cells.

    PubMed

    Pierce, Jacqueline B; Chafe, Shawn C; Eswara, Manoja B K; van der Merwe, George; Mangroo, Dev

    2014-01-01

    Nuclear-cytoplasmic tRNA transport involves multiple pathways that are segregated by the involvement of distinct proteins. The tRNA export process begins in the nucleolus, where the functionality of newly produced tRNAs are tested by aminoacylation, and ends with the delivery of the exported aminoacyl tRNAs to the eukaryotic elongation factor eEF-1A for utilization in protein synthesis in the cytoplasm. Recent studies have identified a number of proteins that participate in nuclear tRNA export in both yeast and mammals. However, genetic and biochemical evidence suggest that additional components, which have yet to be identified, also participate in nuclear-cytoplasmic tRNA trafficking. Here we review key strategies that have led to the identification and characterization of proteins that are involved in the nuclear tRNA export process in yeasts and mammals. The approaches described will greatly facilitate the identification and delineation of the roles of new proteins involved in nuclear export of tRNAs to the cytoplasm.

  6. Factors that influence T box riboswitch efficacy and tRNA affinity.

    PubMed

    Zeng, C; Zhou, S; Bergmeier, S C; Hines, J V

    2015-09-01

    The T box riboswitch is an intriguing potential target for antibacterial drug discovery. Found primarily in Gram-positive bacteria, the riboswitch regulates gene expression by selectively responding to uncharged tRNA to control transcription readthrough. Polyamines and molecular crowding are known to specifically affect RNA function, but their effect on T box riboswitch efficacy and tRNA affinity have not been fully characterized. A fluorescence-monitored in vitro transcription assay was developed to readily quantify these molecular interactions and to provide a moderate-throughput functional assay for a comprehensive drug discovery screening cascade. The polyamine spermidine specifically enhanced T box riboswitch readthrough efficacy with an EC50 = 0.58 mM independent of tRNA binding. Molecular crowding, simulated by the addition of polyethylene glycol, had no effect on tRNA affinity for the riboswitch, but did reduce the efficacy of tRNA-induced readthrough. These results indicate that the T box riboswitch tRNA affinity and readthrough efficacy are intricately modulated by environmental factors. PMID:26220520

  7. The effect of tRNA levels on decoding times of mRNA codons

    PubMed Central

    Dana, Alexandra; Tuller, Tamir

    2014-01-01

    The possible effect of transfer ribonucleic acid (tRNA) concentrations on codons decoding time is a fundamental biomedical research question; however, due to a large number of variables affecting this process and the non-direct relation between them, a conclusive answer to this question has eluded so far researchers in the field. In this study, we perform a novel analysis of the ribosome profiling data of four organisms which enables ranking the decoding times of different codons while filtering translational phenomena such as experimental biases, extreme ribosomal pauses and ribosome traffic jams. Based on this filtering, we show for the first time that there is a significant correlation between tRNA concentrations and the codons estimated decoding time both in prokaryotes and in eukaryotes in natural conditions (−0.38 to −0.66, all P values <0.006); in addition, we show that when considering tRNA concentrations, codons decoding times are not correlated with aminoacyl-tRNA levels. The reported results support the conjecture that translation efficiency is directly influenced by the tRNA levels in the cell. Thus, they should help to understand the evolution of synonymous aspects of coding sequences via the adaptation of their codons to the tRNA pool. PMID:25056313

  8. Of P and Z: mitochondrial tRNA processing enzymes.

    PubMed

    Rossmanith, Walter

    2012-01-01

    Mitochondrial tRNAs are generally synthesized as part of polycistronic transcripts. Release of tRNAs from these precursors is thus not only required to produce functional adaptors for translation, but also responsible for the maturation of other mitochondrial RNA species. Cleavage of mitochondrial tRNAs appears to be exclusively accomplished by endonucleases. 5'-end maturation in the mitochondria of different Eukarya is achieved by various kinds of RNase P, representing the full range of diversity found in this enzyme family. While ribonucleoprotein enzymes with RNA components of bacterial-like appearance are found in a few unrelated protists, algae, and fungi, highly degenerate RNAs of dramatic size variability are found in the mitochondria of many fungi. The majority of mitochondrial RNase P enzymes, however, appear to be pure protein enzymes. Human mitochondrial RNase P, the first to be identified and possibly the prototype of all animal mitochondrial RNases P, is composed of three proteins. Homologs of its nuclease subunit MRPP3/PRORP, are also found in plants, algae and several protists, where they are apparently responsible for RNase P activity in mitochondria (and beyond) without the help of extra subunits. The diversity of RNase P enzymes is contrasted by the uniformity of mitochondrial RNases Z, which are responsible for 3'-end processing. Only the long form of RNase Z, which is restricted to eukarya, is found in mitochondria, even when an additional short form is present in the same organism. Mitochondrial tRNA processing thus appears dominated by new, eukaryal inventions rather than bacterial heritage. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

  9. Characterization of a Salmonella typhimurium hisU mutant defective in tRNA precursor processing.

    PubMed Central

    Bossi, L; Ciampi, M S; Cortese, R

    1978-01-01

    The DA11 mutant of Salmonella typhimurium, originally isolated as derepressed for the histidine operon, carries a temperature-dependent alteration in a nucleolytic enzyme specifically involved in the maturation of tRNA. As a consequence of this alteration, no detectable synthesis of any mature tRNA species occurs in DA11 upon shift at 43 degrees C, whereas many tRNA precursors, whose sizes range between 80 and 750 nucleotides, do accumulate. Kinetic studies on the synthesis and processing of these maturation intermediates show that these molecules represent different stages in the maturation pathway, most of them being the products of previous nucleolytic events. These RNA molecules are in vivo substrates of methylation and thiolation enzymes and can be cleaved in vitro to 4S RNA by wild-type but not by DA11 cell-free extract. Evidence is presented that DA11 is very probably a ribonuclease P mutant. Images PMID:350829

  10. Dynamical analysis of tRNA Gln-GlnRS complex using normal mode calculation

    NASA Astrophysics Data System (ADS)

    Nakamura, Shugo; Ikeguchi, Mitsunori; Shimizu, Kentaro

    2003-04-01

    We applied normal mode calculation in internal coordinates to a complex of glutamine transfer RNA (tRNA Gln) and glutaminyl-tRNA synthetase (GlnRS). Calculated deviations of atoms agreed well with those obtained from X-ray data. The differences of motions corresponding to low mode frequencies between the free state and the complex state were analyzed. For GlnRS, many motions in the free state were conserved in the complex state, while the dynamics of tRNA Gln was largely affected by the complex formation. Superimposed images of the conserved and non-conserved motions of tRNA Gln clearly indicated the restricted direction of motions in the complex.

  11. Saturation of recognition elements blocks evolution of new tRNA identities

    PubMed Central

    Saint-Léger, Adélaïde; Bello, Carla; Dans, Pablo D.; Torres, Adrian Gabriel; Novoa, Eva Maria; Camacho, Noelia; Orozco, Modesto; Kondrashov, Fyodor A.; Ribas de Pouplana, Lluís

    2016-01-01

    Understanding the principles that led to the current complexity of the genetic code is a central question in evolution. Expansion of the genetic code required the selection of new transfer RNAs (tRNAs) with specific recognition signals that allowed them to be matured, modified, aminoacylated, and processed by the ribosome without compromising the fidelity or efficiency of protein synthesis. We show that saturation of recognition signals blocks the emergence of new tRNA identities and that the rate of nucleotide substitutions in tRNAs is higher in species with fewer tRNA genes. We propose that the growth of the genetic code stalled because a limit was reached in the number of identity elements that can be effectively used in the tRNA structure. PMID:27386510

  12. A mitochondrial tRNA aspartate mutation causing isolated mitochondrial myopathy.

    PubMed

    Seneca, Sara; Goemans, Nathalie; Van Coster, Rudy; Givron, Patrice; Reybrouck, Tony; Sciot, Raf; Meulemans, Ann; Smet, Joel; Van Hove, Johan L K

    2005-08-30

    Several mutations in mitochondrial transfer RNA (tRNA) genes can cause mitochondrial myopathy. We describe a young girl who presented with pronounced exercise intolerance. The anaerobic threshold and the maximal oxygen consumption were decreased. She had decreased complex I and IV enzyme activity and ragged red fibers on muscle biopsy. An A to G transition at nucleotide position 7526 in tRNA Aspartate (tRNA(Asp)) gene was heteroplasmic in several of the patient's tissues. We were unable to detect the mutation in muscle tissue from the patient's mother. This case adds a new genetic etiology for mitochondrial myopathy. It also illustrates for patients with combined deficiency of the complex I and IV enzyme activity the value of sequencing in the affected tissue muscle, and not only in blood, all mitochondrial tRNA genes including those not commonly affected, such as in this case mt tRNA(Asp).

  13. Decoding in Candidatus Riesia pediculicola, close to a minimal tRNA modification set?

    PubMed Central

    de Crécy-Lagard, Valérie; Marck, Christian; Grosjean, Henri

    2012-01-01

    A comparative genomic analysis of the recently sequenced human body louse unicellular endosymbiont Candidatus Riesia pediculicola with a reduced genome (582 Kb), revealed that it is the only known organism that might have lost all post-transcriptional base and ribose modifications of the tRNA body, retaining only modifications of the anticodon-stem-loop essential for mRNA decoding. Such a minimal tRNA modification set was not observed in other insect symbionts or in parasitic unicellular bacteria, such as Mycoplasma genitalium (580 Kb), that have also evolved by considerably reducing their genomes. This could be an example of a minimal tRNA modification set required for life, a question that has been at the center of the field for many years, especially for understanding the emergence and evolution of the genetic code. PMID:23308034

  14. From Prebiotics to Probiotics: The Evolution and Functions of tRNA Modifications

    PubMed Central

    McKenney, Katherine M.; Alfonzo, Juan D.

    2016-01-01

    All nucleic acids in cells are subject to post-transcriptional chemical modifications. These are catalyzed by a myriad of enzymes with exquisite specificity and that utilize an often-exotic array of chemical substrates. In no molecule are modifications more prevalent than in transfer RNAs. In the present document, we will attempt to take a chemical rollercoaster ride from prebiotic times to the present, with nucleoside modifications as key players and tRNA as the centerpiece that drove the evolution of biological systems to where we are today. These ideas will be put forth while touching on several examples of tRNA modification enzymes and their modus operandi in cells. In passing, we submit that the choice of tRNA is not a whimsical one but rather highlights its critical function as an essential invention for the evolution of protein enzymes. PMID:26985907

  15. Metazoan tRNA introns generate stable circular RNAs in vivo

    PubMed Central

    Lu, Zhipeng; Filonov, Grigory S.; Noto, John J.; Schmidt, Casey A.; Hatkevich, Talia L.; Wen, Ying; Jaffrey, Samie R.; Matera, A. Gregory

    2015-01-01

    We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating “designer” circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. PMID:26194134

  16. Saturation of recognition elements blocks evolution of new tRNA identities.

    PubMed

    Saint-Léger, Adélaïde; Bello, Carla; Dans, Pablo D; Torres, Adrian Gabriel; Novoa, Eva Maria; Camacho, Noelia; Orozco, Modesto; Kondrashov, Fyodor A; Ribas de Pouplana, Lluís

    2016-04-01

    Understanding the principles that led to the current complexity of the genetic code is a central question in evolution. Expansion of the genetic code required the selection of new transfer RNAs (tRNAs) with specific recognition signals that allowed them to be matured, modified, aminoacylated, and processed by the ribosome without compromising the fidelity or efficiency of protein synthesis. We show that saturation of recognition signals blocks the emergence of new tRNA identities and that the rate of nucleotide substitutions in tRNAs is higher in species with fewer tRNA genes. We propose that the growth of the genetic code stalled because a limit was reached in the number of identity elements that can be effectively used in the tRNA structure. PMID:27386510

  17. Identification of highly-disrupted tRNA genes in nuclear genome of the red alga, Cyanidioschyzon merolae 10D

    PubMed Central

    Soma, Akiko; Sugahara, Junichi; Onodera, Akinori; Yachie, Nozomu; Kanai, Akio; Watanabe, Satoru; Yoshikawa, Hirofumi; Ohnuma, Mio; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi; Sekine, Yasuhiko

    2013-01-01

    The limited locations of tRNA introns are crucial for eukaryal tRNA-splicing endonuclease recognition. However, our analysis of the nuclear genome of an early-diverged red alga, Cyanidioschyzon merolae, demonstrated the first evidence of nuclear-encoded tRNA genes that contain ectopic and/or multiple introns. Some genes exhibited both intronic and permuted structures in which the 3′-half of the tRNA coding sequence lies upstream of the 5′-half, and an intron is inserted into either half. These highly disrupted tRNA genes, which account for 63% of all nuclear tRNA genes, are expressed via the orderly and sequential processing of bulge-helix-bulge (BHB) motifs at intron-exon junctions and termini of permuted tRNA precursors, probably by a C. merolae tRNA-splicing endonuclease with an unidentified subunit architecture. The results revealed a considerable diversity in eukaryal tRNA intron properties and endonuclease architectures, which will help to elucidate the acquisition mechanism of the BHB-mediated disrupted tRNA genes. PMID:23900518

  18. An evolutionary approach uncovers a diverse response of tRNA 2-thiolation to elevated temperatures in yeast

    PubMed Central

    Alings, Fiona; Sarin, L. Peter; Fufezan, Christian; Drexler, Hannes C.A.

    2015-01-01

    Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s2U34) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s2U34 nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s2U34 modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms. PMID:25505025

  19. The human decorin gene: Intron-exon organization, discovery of two alternatively spliced exons in the 5[prime] untralsated region, and mapping of the gene to chromosome 12q23

    SciTech Connect

    Danielson, K.G.; Fazzio, A.; Cohen, I.; Cannizzaro, L.A.; Eichstetter, I.; Iozzo, R.V. )

    1993-01-01

    Decorin is a chondroitin/dermatan sulfate proteoglycan expressed by most vascular and avascular connective tissues and, because of its ability to interact with collagen and growth factors, has been implicated in the control of matrix assembly and cellular growth. To understand the molecular mechanisms involved in regulating its tissue expression, we have isolated a number of genomic clones encoding the complete decorin gene. The human decorin gene spans over 38 kb of continuous DNA sequence and contains eight exons and very large introns, two of which are 5.4 and > 13.2 kb. We have discovered two alternatively spliced leader exons, exons Ia and Ib, in the 5[prime] untranslated region. These exons were identified by cloning and sequencing cDNAs obtained by polymerase chain reaction amplification of a fibroblast cDNA library. Using Northern blotting or reverse transcriptase PCR, we detected the two leader exons in a variety of mRNAs isolated from human cell lines and tissues. Interestingly, sequences highly (74-87%) homologous to exons Ia and lb are found in the 5[prime]untranslated region of avian and bovine decorin, respectively. This high degree of conservation among species suggests regulatory functions for these leader exons. In the 3' untranslated region there are several polyadenylation sites, and at least two of these sites could give rise to the transcripts of [approx]1.6 and [approx]1.9 kb, typically detected in a variety of tissues and cells. Using a genomic clone as the labeled probe and in situ hybridization of human metaphase chromosomes, we have mapped the decorin gene to the discrete region of human chromosome 12q23. This sturdy provides the molecular basis for discerning the transcriptional control of the decorin gene and offers the opportunity to investigate genetic disorders linked to this important human gene. 57 refs., 11 figs., 3 tabs.

  20. Nucleotides of the tRNA D-stem that play an important role in nuclear-tRNA export in Saccharomyces cerevisiae.

    PubMed

    Cleary, J D; Mangroo, D

    2000-04-01

    Nuclear export of tRNA in Saccharomyces cerevisiae involves Los1p and Arc1p. Los1p facilitates tRNA translocation across the nuclear pore complex whereas Arc1p plays a role in delivering some species of tRNA exiting the nucleus to their cognate aminoacyl-tRNA synthetases. Here, we show that mutations of C11 and G24 of the D-stem of the yeast tyrosine amber-suppressor tRNA have different effects on nuclear export of the tRNA. Changing G24 had no effect on export of the tRNA to the cytoplasm. In contrast, mutating C11 resulted in nuclear retention of the tRNA. Nuclear retention of the tRNA mutants was not due to lack of processing, since only the mature forms of the tRNA mutants were found. The fact that mutations of G24 did not affect export of the tRNA also indicates that the effect of mutating C11 is not due to gross alteration of the tertiary structure resulting from disruption of the C11/G24 base pair. Expression of Los1p and the mammalian tRNA export receptor exportin-t rescued nuclear export of the tRNA with changes at position 11. The export-defective mutations of the tRNA mutants were suppressed by introducing the complementary nucleotides at position 24. Taken together, these findings suggest that C11 is important for binding of the tRNA to the export receptor, and that this binding is influenced by the conformation of the base. Finally, the export-defective tRNA mutants described can be used as reporters to identify eukaryotic proteins involved in the nuclear-tRNA export process, and characterize the molecular interactions between known receptors and the tRNA substrate.

  1. tRNA's wobble decoding of the genome: 40 years of modification.

    PubMed

    Agris, Paul F; Vendeix, Franck A P; Graham, William D

    2007-02-01

    The genetic code is degenerate, in that 20 amino acids are encoded by 61 triplet codes. In 1966, Francis Crick hypothesized that the cell's limited number of tRNAs decoded the genome by recognizing more than one codon. The ambiguity of that recognition resided in the third base-pair, giving rise to the Wobble Hypothesis. Post-transcriptional modifications at tRNA's wobble position 34, especially modifications of uridine 34, enable wobble to occur. The Modified Wobble Hypothesis proposed in 1991 that specific modifications of a tRNA wobble nucleoside shape the anticodon architecture in such a manner that interactions were restricted to the complementary base plus a single wobble pairing for amino acids with twofold degenerate codons. However, chemically different modifications at position 34 would expand the ability of a tRNA to read three or even four of the fourfold degenerate codons. One foundation of Crick's Wobble Hypothesis was that a near-constant geometry of canonical base-pairing be maintained in forming all three base-pairs between the tRNA anticodon and mRNA codon on the ribosome. In accepting an aminoacyl-tRNA, the ribosome requires maintenance of a specific geometry for the anticodon-codon base-pairing. However, it is the post-transcriptional modifications at tRNA wobble position 34 and purine 37, 3'-adjacent to the anticodon, that pre-structure the anticodon domain to ensure the correct codon binding. The modifications create both the architecture and the stability needed for decoding through restraints on anticodon stereochemistry and conformational space, and through selective hydrogen bonding. A physicochemical understanding of modified nucleoside contributions to the tRNA anticodon domain architecture and its decoding of the genome has advanced RNA world evolutionary theory, the principles of RNA chemistry, and the application of this knowledge to the introduction of new amino acids to proteins.

  2. A voltage-gated pore for translocation of tRNA

    SciTech Connect

    Koley, Sandip; Adhya, Samit

    2013-09-13

    Highlights: •A tRNA translocating complex was assembled from purified proteins. •The complex translocates tRNA at a membrane potential of ∼60 mV. •Translocation requires Cys and His residues in the Fe–S center of RIC6 subunit. -- Abstract: Very little is known about how nucleic acids are translocated across membranes. The multi-subunit RNA Import Complex (RIC) from mitochondria of the kinetoplastid protozoon Leishmania tropica induces translocation of tRNAs across artificial or natural membranes, but the nature of the translocation pore remains unknown. We show that subunits RIC6 and RIC9 assemble on the membrane in presence of subunit RIC4A to form complex R3. Atomic Force Microscopy of R3 revealed particles with an asymmetric surface groove of ∼20 nm rim diameter and ∼1 nm depth. R3 induced translocation of tRNA into liposomes when the pH of the medium was lowered to ∼6 in the absence of ATP. R3-mediated tRNA translocation could also be induced at neutral pH by a K{sup +} diffusion potential with an optimum of 60–70 mV. Point mutations in the Cys{sub 2}–His{sub 2} Fe-binding motif of RIC6, which is homologous to the respiratory Complex III Fe–S protein, abrogated import induced by low pH but not by K{sup +} diffusion potential. These results indicate that the R3 complex forms a pore that is gated by a proton-generated membrane potential and that the Fe–S binding region of RIC6 has a role in proton translocation. The tRNA import complex of L. tropica thus contains a novel macromolecular channel distinct from the mitochondrial protein import pore that is apparently involved in tRNA import in some species.

  3. Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10

    PubMed Central

    Swinehart, William E.; Henderson, Jeremy C.; Jackman, Jane E.

    2013-01-01

    N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m1G9 modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m1G9 in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m1G9 methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m1G9 containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing. PMID:23793893

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

    PubMed

    Hori, Hiroyuki; Terui, Yusuke; Nakamoto, Chisato; Iwashita, Chikako; Ochi, Anna; Watanabe, Kazunori; Oshima, Tairo

    2016-05-01

    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. PMID:26721905

  5. In vitro tRNA methylation assay with the Entamoeba histolytica DNA and tRNA methyltransferase Dnmt2 (Ehmeth) enzyme.

    PubMed

    Tovy, Ayala; Hofmann, Benjamin; Helm, Mark; Ankri, Serge

    2010-01-01

    Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, Dictyostelium discoideum and Drosophila. Our recent work has shown that Ehmeth methylates tRNA(Asp), and this finding indicates that this enzyme has a dual DNA/tRNA(Asp) methyltransferase activity. This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity. PMID:21048666

  6. tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry.

    PubMed

    Carter, Charles W; Wolfenden, Richard

    2016-01-01

    The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology.

  7. Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes

    PubMed Central

    Adir, Idan; Dahan, Orna; Broday, Limor; Pilpel, Yitzhak; Rechavi, Oded

    2016-01-01

    Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal’s lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene’s promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species. PMID:27560950

  8. Transfer RNA: From pioneering crystallographic studies to contemporary tRNA biology.

    PubMed

    Fernández-Millán, Pablo; Schelcher, Cédric; Chihade, Joseph; Masquida, Benoît; Giegé, Philippe; Sauter, Claude

    2016-07-15

    Transfer RNAs (tRNAs) play a key role in protein synthesis as adaptor molecules between messenger RNA and protein sequences on the ribosome. Their discovery in the early sixties provoked a worldwide infatuation with the study of their architecture and their function in the decoding of genetic information. tRNAs are also emblematic molecules in crystallography: the determination of the first tRNA crystal structures represented a milestone in structural biology and tRNAs were for a long period the sole source of information on RNA folding, architecture, and post-transcriptional modifications. Crystallographic data on tRNAs in complex with aminoacyl-tRNA synthetases (aaRSs) also provided the first insight into protein:RNA interactions. Beyond the translation process and the history of structural investigations on tRNA, this review also illustrates the renewal of tRNA biology with the discovery of a growing number of tRNA partners in the cell, the involvement of tRNAs in a variety of regulatory and metabolic pathways, and emerging applications in biotechnology and synthetic biology.

  9. Dependence of RelA-mediated (p)ppGpp formation on tRNA identity.

    PubMed

    Payoe, Roshani; Fahlman, Richard P

    2011-04-19

    The bacterial stringent response is a cellular response to amino acid limitations and is characterized by the accumulation of the alarmone polyphosphate guanosine ((p)ppGpp). A key molecular event leading to (p)ppGpp synthesis is the binding of a deacylated tRNA to the vacant A-Site of a ribosome. The resulting ribosomal complex is recognized by and activates RelA, the (p)ppGpp synthetase. Activated RelA catalyzes (p)ppGpp formation until the deacylated tRNA passively dissociates from the ribosomal A-Site. In this report, we have investigated a novel role for the identity of A-Site bound tRNA in RelA-mediated (p)ppGpp synthesis. A comparison in the stimulation of RelA activity was made using ribosome complexes with either a tightly or weakly binding deacylated tRNA occupying the A-Site. In vitro analysis reveals that ribosome complexes formed with tight binding tRNA(Val) stimulate RelA activity at lower concentrations than that required for ribosome complexes formed with the weaker binding tRNA(Phe). The data suggest that the recovery from the stringent response may be dependent on the identity of the amino acid that was initially limiting for the bacteria.

  10. Biochemical and Structures Studies of tRNA Modificaton and Repair Enzymes

    ERIC Educational Resources Information Center

    Zhou, Chun

    2009-01-01

    RNA hypermodifications near the anticodon of tRNA are fundamental for the efficiency and fidelity of protein synthesis. Dimethylallyltransferase (DMATase) catalyzes transfer of a dimethylallyl moiety from dimethylallyl pyrophosphate to N6 of A37 in certain tRNAs. We first determined the crystal structures of "Pseudomonas aeruginosa" DMATase.…

  11. Evaluation of tRNA Gene PCR for Identification of Mollicutes

    PubMed Central

    Stakenborg, Tim; Vicca, Jo; Verhelst, Rita; Butaye, Patrick; Maes, Dominiek; Naessens, Anne; Claeys, Geert; De Ganck, Catharine; Haesebrouck, Freddy; Vaneechoutte, Mario

    2005-01-01

    We evaluated the applicability of tRNA gene PCR in combination with fluorescent capillary electrophoresis with an ABI310 genetic analyzer (Applied Biosystems, Calif.) for the identification of different mollicute species. A total of 103 strains and DNA extracts of 30 different species belonging to the genera Acholeplasma, Mycoplasma, and Ureaplasma were studied. Reproducible peak profiles were generated for all samples, except for one M. genitalium isolate, the three M. gallisepticum isolates, and 8 of the 24 Ureaplasma cultures, where no amplification could be obtained. Clustering revealed numerous discrepancies compared to the identifications that had been previously obtained by means of biochemical and serological tests. Final identification was obtained by 16S rRNA gene amplification followed by sequence analysis and/or restriction digestion. This confirmed the identification obtained by tRNA gene PCR in all cases. Seven samples yielded an unexpected tRNA gene PCR profile. Sequence analysis of the 16S rRNA genes showed that six of these samples were mixed and that one had a unique sequence that did not match any of the published sequences, pointing to the existence of a not-yet-described species. In conclusion, we found tRNA gene PCR to be a rapid and discriminatory method to correctly identify a large collection of different species of the class of Mollicutes and to recognize not-yet-described groups. PMID:16145107

  12. tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry.

    PubMed

    Carter, Charles W; Wolfenden, Richard

    2016-01-01

    The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology. PMID:26595350

  13. N6-methyladenosine in mRNA disrupts tRNA selection and translation elongation dynamics

    PubMed Central

    Choi, Junhong; Ieong, Ka-Weng; Demirci, Hasan; Chen, Jin; Petrov, Alexey; Prabhakar, Arjun; O'Leary, Seán E.; Dominissini, Dan; Rechavi, Gideon; Soltis, S. Michael; Ehrenberg, Måns

    2016-01-01

    N6-methylation of adenosine (m6A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m6A in mRNA decoding. Although m6A base pairs with uridine during decoding as shown by x-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements employing an Escherichia coli translation system revealed that m6A modification of mRNA can act as a barrier to tRNA accommodation and translation elongation. The interaction between an m6A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, as delay in tRNA accommodation depends on the position and context of m6A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics. PMID:26751643

  14. Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy.

    PubMed

    Fischer, Niels; Konevega, Andrey L; Wintermeyer, Wolfgang; Rodnina, Marina V; Stark, Holger

    2010-07-15

    The translocation step of protein synthesis entails large-scale rearrangements of the ribosome-transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during translocation by time-resolved single-particle electron cryomicroscopy (cryo-EM). Unbiased computational sorting of cryo-EM images yielded 50 distinct three-dimensional reconstructions, showing the tRNAs in classical, hybrid and various novel intermediate states that provide trajectories and kinetic information about tRNA movement through the ribosome. The structures indicate how tRNA movement is coupled with global and local conformational changes of the ribosome, in particular of the head and body of the small ribosomal subunit, and show that dynamic interactions between tRNAs and ribosomal residues confine the path of the tRNAs through the ribosome. The temperature dependence of ribosome dynamics reveals a surprisingly flat energy landscape of conformational variations at physiological temperature. The ribosome functions as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to directed movement.

  15. Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes.

    PubMed

    Sagi, Dror; Rak, Roni; Gingold, Hila; Adir, Idan; Maayan, Gadi; Dahan, Orna; Broday, Limor; Pilpel, Yitzhak; Rechavi, Oded

    2016-08-01

    Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal's lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene's promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species. PMID:27560950

  16. CLP1 Founder Mutation Links tRNA Splicing and Maturation to Cerebellar Development and Neurodegeneration

    PubMed Central

    Schaffer, Ashleigh E.; Eggens, Veerle R.C.; Caglayan, Ahmet Okay; Reuter, Miriam S.; Scott, Eric; Coufal, Nicole G.; Silhavy, Jennifer L.; Xue, Yuanchao; Kayserili, Hulya; Yasuno, Katsuhito; Rosti, Rasim Ozgur; Abdellateef, Mostafa; Caglar, Caner; Kasher, Paul R.; Cazemier, J. Leonie; Weterman, Marian A.; Cantagrel, Vincent; Cai, Na; Zweier, Christiane; Altunoglu, Umut; Satkin, N. Bilge; Aktar, Fesih; Tuysuz, Beyhan; Yalcinkaya, Cengiz; Caksen, Huseyin; Bilguvar, Kaya; Fu, Xiang-Dong; Trotta, Christopher; Gabriel, Stacey; Reis, André; Gunel, Murat; Baas, Frank; Gleeson, Joseph G.

    2014-01-01

    SUMMARY Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of over 2000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild type but not mutant human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data links tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans. PMID:24766810

  17. Quantitative analysis of tRNA modifications by HPLC-coupled mass spectrometry

    PubMed Central

    Su, Dan; Chan, Clement T.Y.; Gu, Chen; Lim, Kok Seong; Chionh, Yok Hian; McBee, Megan E.; Russell, Brandon S.; Babu, I. Ramesh; Begley, Thomas J.; Dedon, Peter C.

    2015-01-01

    Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions, and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress response mechanism involving selective translation of codon-biased mRNA for critical proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-coupled mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA or other RNA species. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS using dynamic multiple reaction monitoring. This approach enables quantification of modified ribonucleosides in several micrograms of tRNA, or other RNA, in a 15-minute LC-MS run. By comparison, traditional methods for detecting modified ribonucleosides are labor and time intensive, require larger RNA quantities, are modification-specific, or require radioactive labeling. PMID:24625781

  18. Elongation factor 4 remodels the A-site tRNA on the ribosome

    PubMed Central

    Gagnon, Matthieu G.; Lin, Jinzhong; Steitz, Thomas A.

    2016-01-01

    During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant. PMID:27092003

  19. In vitro import of a nuclearly encoded tRNA into mitochondria of Solanum tuberosum.

    PubMed

    Delage, Ludovic; Dietrich, André; Cosset, Anne; Maréchal-Drouard, Laurence

    2003-06-01

    Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNA(Ala), tRNA(Phe), and tRNA(Met-e) with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNA(Ala) than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNA(Ala) is naturally imported into potato mitochondria whereas tRNA(Phe) and tRNA(Met-e) are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential.

  20. Pentamidine binds to tRNA through non-specific hydrophobic interactions and inhibits aminoacylation and translation.

    PubMed

    Sun, Tao; Zhang, Yi

    2008-03-01

    The selective and potent inhibition of mitochondrial translation in Saccharomyces cerevisiae by pentamidine suggests a novel antimicrobial action for this drug. Electrophoresis mobility shift assay, T1 ribonuclease footprinting, hydroxyl radical footprinting and isothermal titration calorimetry collectively demonstrated that pentamidine non-specifically binds to two distinct classes of sites on tRNA. The binding was driven by favorable entropy changes indicative of a large hydrophobic interaction, suggesting that the aromatic rings of pentamidine are inserted into the stacked base pairs of tRNA helices. Pentamidine binding disrupts the tRNA secondary structure and masks the anticodon loop in the tertiary structure. Consistently, we showed that pentamidine specifically inhibits tRNA aminoacylation but not the cognate amino acid adenylation. Pentamidine inhibited protein translation in vitro with an EC(50) equivalent to that binds to tRNA and inhibits tRNA aminoacylation in vitro, but drastically higher than that inhibits translation in vivo, supporting the established notion that the antimicrobial activity of pentamidine is largely due to its selective accumulation by the pathogen rather than by the host cell. Therefore, interrupting tRNA aminoacylation by the entropy-driven non-specific binding is an important mechanism of pentamidine in inhibiting protein translation, providing new insights into the development of antimicrobial drugs.

  1. Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

    SciTech Connect

    Ogawa, Tetsuhiro Shimizu, Ayano; Takahashi, Kazutoshi; Hidaka, Makoto; Masaki, Haruhiko

    2014-08-15

    Highlights: • MTS-tagged ribonuclease was translocated successfully to the mitochondrial matrix. • MTS-tagged ribonuclease cleaved mt tRNA and reduced COX activity. • Easy and reproducible method of inducing mt tRNA dysfunction. - Abstract: Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ{sup 0} cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.

  2. Yeast tRNA3Leu gene transcribed and spliced in a HeLa cell extract.

    PubMed Central

    Standring, D N; Venegas, A; Rutter, W J

    1981-01-01

    A cloned yeast tRNA3Leu gene containing a 33-base intervening sequence (IVS) is selectively transcribed by a soluble extract from HeLa cells. The 130-nucleotide tRNA3Leu precursor RNA formed is colinear with the gene and contains approximately 4 leader nucleotides and up to 9 trailer nucleotides. The IVS is accurately and efficiently removed by an endogenous HeLa excision-ligase activity to yield the spliced tRNA, the free IVS, and the half-tRNA intermediates. The splicing reaction occurs without prior 5' and 3' maturation of the precursor but, with this exception, this pattern of synthesis and subsequent maturation of the tRNA3Leu precursor conforms to the scheme for tRNA biosynthesis deduced for the xenopus system. Indeed, the two systems utilize similar or identical tRNA3Leu precursors. Our results stress the extraordinary conservation of tRNA biosynthesis in eukaryotes and demonstrate that a HeLa extract provides a useful system for investigating this process. Images PMID:6796956

  3. Evolution meets disease: penetrance and functional epistasis of mitochondrial tRNA mutations.

    PubMed

    Moreno-Loshuertos, Raquel; Ferrín, Gustavo; Acín-Pérez, Rebeca; Gallardo, M Esther; Viscomi, Carlo; Pérez-Martos, Acisclo; Zeviani, Massimo; Fernández-Silva, Patricio; Enríquez, José Antonio

    2011-04-01

    About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m.4290T>C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNA(Ile) that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both. PMID:21533077

  4. Degradation of 4,4{prime}-Dichlorobiphenyl, 3,3{prime}, 4,4{prime}-Tetrachlorobiphenyl, and 2,2{prime},4,4{prime},5,5{prime}-Hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium

    SciTech Connect

    Dietrich, D.; Lamar, R.; Hickey, W.J.

    1995-11-01

    The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4{prime}- dichlorobiphenyl [DCB], 3,3{prime},4,4{prime}-tetrachlorobiphenyl, and 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, {sup 14}C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the {sup 14}C was determined to be biomass bound. Results from a time course study done with 4,4{prime}-[{sup 14}C]DCB to examine {sup 14}C partitioning dynamics indicated that the biomass-bound {sup 14}C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4{prime}-[{sup 14}C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process. 23 refs., 4 figs., 2 tabs.

  5. Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication

    PubMed Central

    Lund, Anders H.; Duch, Mogens; Pedersen, Finn Skou

    2000-01-01

    Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNAPro molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retroviruses have revealed evidence of molecular adaptation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3′-end of tRNAPro, we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNAArg(CCU), tRNAPhe(GAA) and a hitherto unknown human tRNASer(CGA). PMID:10637332

  6. The Temperature Sensitivity of a Mutation in the Essential tRNA Modification Enzyme tRNA Methyltransferase D (TrmD)*

    PubMed Central

    Masuda, Isao; Sakaguchi, Reiko; Liu, Cuiping; Gamper, Howard; Hou, Ya-Ming

    2013-01-01

    Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m1G37 on the 3′ side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation. PMID:23986443

  7. The m1A(58) modification in eubacterial tRNA: An overview of tRNA recognition and mechanism of catalysis by TrmI.

    PubMed

    Dégut, Clément; Ponchon, Luc; Folly-Klan, Marcia; Barraud, Pierre; Tisné, Carine

    2016-03-01

    The enzymes of the TrmI family catalyze the formation of the m(1)A58 modification in tRNA. We previously solved the crystal structure of the Thermus thermophilus enzyme and conducted a biophysical study to characterize the interaction between TrmI and tRNA. TrmI enzymes are active as a tetramer and up to two tRNAs can bind to TrmI simultaneously. In this paper, we present the structures of two TrmI mutants (D170A and Y78A). These residues are conserved in the active site of TrmIs and their mutations result in a dramatic alteration of TrmI activity. Both structures of TrmI mutants revealed the flexibility of the N-terminal domain that is probably important to bind tRNA. The structure of TrmI Y78A catalytic domain is unmodified regarding the binding of the SAM co-factor and the conformation of residues potentially interacting with the substrate adenine. This structure reinforces the previously proposed role of Y78, i.e. stabilize the conformation of the A58 ribose needed to hold the adenosine in the active site. The structure of the D170A mutant shows a flexible active site with one loop occupying in part the place of the co-factor and the second loop moving at the entrance to the active site. This structure and recent data confirms the central role of D170 residue binding the amino moiety of SAM and the exocyclic amino group of adenine. Possible mechanisms for methyl transfer are then discussed.

  8. Substrate tRNA recognition mechanism of a multisite-specific tRNA methyltransferase, Aquifex aeolicus Trm1, based on the X-ray crystal structure.

    PubMed

    Awai, Takako; Ochi, Anna; Ihsanawati; Sengoku, Toru; Hirata, Akira; Bessho, Yoshitaka; Yokoyama, Shigeyuki; Hori, Hiroyuki

    2011-10-01

    Archaeal and eukaryotic tRNA (N(2),N(2)-guanine)-dimethyltransferase (Trm1) produces N(2),N(2)-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of A. aeolicus Trm1. The N-terminal domain is a typical catalytic domain of S-adenosyl-l-methionine-dependent methyltransferases. On the basis of the crystal structure and amino acid sequence alignment, we prepared 30 mutant Trm1 proteins. These mutant proteins clarified residues important for S-adenosyl-l-methionine binding and enabled us to propose a hypothetical reaction mechanism. Furthermore, the tRNA-binding site was also elucidated by methyl transfer assay and gel mobility shift assay. The electrostatic potential surface models of A. aeolicus and archaeal Trm1 proteins demonstrated that the distribution of positive charges differs between the two proteins. We constructed a tRNA-docking model, in which the T-arm structure was placed onto the large area of positive charge, which is the expected tRNA-binding site, of A. aeolicus Trm1. In this model, the target G26 base can be placed near the catalytic pocket; however, the nucleotide at position 27 gains closer access to the pocket. Thus, this docking model introduces a rational explanation of the multisite specificity of A. aeolicus Trm1.

  9. Characterization and nucleotide sequence of a chicken gene encoding an opal suppressor tRNA and its flanking DNA segments.

    PubMed Central

    Hatfield, D L; Dudock, B S; Eden, F C

    1983-01-01

    A naturally occurring opal suppressor serine tRNA has been purified from chicken liver and used as a probe to isolate the corresponding gene from a library of chicken DNA in bacteriophage lambda. This minor tRNA is encoded by a single-copy gene that is not part of a tRNA gene cluster. DNA sequence analysis of the gene and its flanking DNA segments shows that the gene is encoded in an 87-base-pair segment without intervening sequences and specifies a tRNA that reads the termination codon UGA. This gene has additional nucleotides in the 5' internal promoter region but has a normal 3' internal promoter sequence and the usual termination signal. Images PMID:6308662

  10. New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA

    PubMed Central

    Kempenaers, Morgane; Roovers, Martine; Oudjama, Yamina; Tkaczuk, Karolina L.; Bujnicki, Janusz M.; Droogmans, Louis

    2010-01-01

    Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N1-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N1 atom of a purine residue is discussed. PMID:20525789

  11. Enhanced dynamics of hydrated tRNA on nanodiamond surfaces: A combined neutron scattering and MD simulation study

    DOE PAGES

    Dhindsa, Gurpreet K.; Bhowmik, Debsindhu; Goswami, Monojoy; O’Neill, Hugh; Mamontov, Eugene; Sumpter, Bobby G.; Hong, Liang; Ganesh, Panchapakesan; Chu, Xiang -qiang

    2016-09-01

    Nontoxic, biocompatible nanodiamonds (ND) have recently been implemented in rational, systematic design of optimal therapeutic use in nanomedicines. However, hydrophilicity of the ND surface strongly influences structure and dynamics of biomolecules that restrict in situ applications of ND. Therefore, fundamental understanding of the impact of hydrophilic ND surface on biomolecules at the molecular level is essential. For tRNA, we observe an enhancement of dynamical behavior in the presence of ND contrary to generally observed slow motion at strongly interacting interfaces. We took advantage of neutron scattering experiments and computer simulations to demonstrate this atypical faster dynamics of tRNA on NDmore » surface. The strong attractive interactions between ND, tRNA, and water give rise to unlike dynamical behavior and structural changes of tRNA in front of ND compared to without ND. As a result, our new findings may provide new design principles for safer, improved drug delivery platforms.« less

  12. Carbonic anhydrase inhibitors with dual-tail moieties to match the hydrophobic and hydrophilic halves of the carbonic anhydrase active site.

    PubMed

    Tanpure, Rajendra P; Ren, Bin; Peat, Thomas S; Bornaghi, Laurent F; Vullo, Daniela; Supuran, Claudiu T; Poulsen, Sally-Ann

    2015-02-12

    We present a new approach to carbonic anhydrase II (CA II) inhibitor design that enables close interrogation of the regions of the CA active site where there is the greatest variability in amino acid residues among the different CA isozymes. By appending dual tail groups onto the par excellence CA inhibitor acetazolamide, compounds that may interact with the distinct hydrophobic and hydrophilic halves of the CA II active site were prepared. The dual-tail combinations selected included (i) two hydrophobic moieties, (ii) two hydrophilic moieties, and (iii) one hydrophobic and one hydrophilic moiety. The CA enzyme inhibition profile as well as the protein X-ray crystal structure of compound 3, comprising one hydrophobic and one hydrophilic tail moiety, in complex with CA II is described. This novel dual-tail approach has provided an enhanced opportunity to more fully exploit interactions with the CA active site by enabling these molecules to interact with the distinct halves of the active site. In addition to the dual-tail compounds, a corresponding set of single-tail derivatives was synthesized, enabling a comparative analysis of the single-tail versus dual-tail compound CA inhibition profile.

  13. Amino acid sequence homology between N- and C-terminal halves of a carbonic anhydrase in Porphyridium purpureum, as deduced from the cloned cDNA.

    PubMed

    Mitsuhashi, S; Miyachi, S

    1996-11-01

    Carbonic anhydrase (CA) from Porphyridium purpureum, a unicellular red alga, was purified >209-fold to a specific activity of 1,147 units/mg protein. cDNA clones for this CA were isolated. The longest clone, comprising 1,960 base pairs, contained an open reading frame which encoded a 571-amino acid polypeptide with a calculated molecular mass of 62,094 Da. The N- and C-terminal halves of the putative mature Porphyridium CA have amino acid sequence homology to each other (>70%) and to other prokaryotic-type CAs. Both regions contain, at equivalent positions, one set of three possible zinc-liganding amino acid residues conserved among prokaryotic-type CAs. CA purified from Porphyridium contained two atoms of zinc per molecule. We propose that the Porphyridium CA has evolved by duplication of an ancestral CA gene followed by the fusion of the duplicated CA gene. The CA truncated into the putative mature form was overexpressed in Escherichia coli, and the expressed protein was active. Clones expressing separately the N- and C-terminal halves of the CA were constructed. CA activity was present in extracts of E. coli cells expressing the N-terminal half, while no detectable activity was found in cells expressing the C-terminal half.

  14. Binding of DNA-binding alkaloids berberine and palmatine to tRNA and comparison to ethidium: Spectroscopic and molecular modeling studies

    NASA Astrophysics Data System (ADS)

    Islam, Md. Maidul; Pandya, Prateek; Chowdhury, Sebanti Roy; Kumar, Surat; Kumar, Gopinatha Suresh

    2008-11-01

    The interaction of two natural protoberberine plant alkaloids berberine and palmatine with tRNA phe was studied using various biophysical techniques and molecular modeling and the data were compared with the binding of the classical DNA intercalator, ethidium. Circular dichroic studies revealed that the tRNA conformation was moderately perturbed on binding of the alkaloids. The cooperative binding of both the alkaloids and ethidium to tRNA was revealed from absorbance and fluorescence studies. Fluorescence quenching studies advanced a conclusion that while berberine and palmatine are partially intercalated, ethidium is fully intercalated on the tRNA molecule. The binding of the alkaloids as well as ethidium stabilized the tRNA melting, and the binding constant evaluated from the averaged optical melting temperature data was in agreement with fluorescence spectral-binding data. Differential scanning calorimetry revealed that the tRNA melting showed three close transitions that were affected on binding of these small molecules. Molecular docking calculations performed showed the preferred regions of binding of these small molecules on the tRNA. Taken together, the results suggest that the binding of the alkaloids berberine and palmatine on the tRNA structure appears to be mostly by partial intercalation while ethidium intercalates fully on the tRNA. These results further advance our knowledge on the molecular aspects on the interaction of these alkaloids to tRNA.

  15. tRNA Modifying Enzymes, NSUN2 and METTL1, Determine Sensitivity to 5-Fluorouracil in HeLa Cells

    PubMed Central

    Hori, Masato; Okada, Kaoru; Yazama, Futoshi; Konishi, Hiroaki; Xiao, Yegui; Qi, Guangying; Shimamoto, Fumio; Ota, Takahide; Temme, Achim; Tatsuka, Masaaki

    2014-01-01

    Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer. PMID:25233213

  16. Translational infidelity-induced protein stress results from a deficiency in Trm9-catalyzed tRNA modifications.

    PubMed

    Patil, Ashish; Chan, Clement T Y; Dyavaiah, Madhu; Rooney, John P; Dedon, Peter C; Begley, Thomas J

    2012-07-01

    Correct codon-anticodon pairing promotes translational fidelity, with these interactions greatly facilitated by modified nucleosides found in tRNA. We hypothesized that wobble uridine modifications catalyzed by tRNA methyltransferase 9 (Trm9) are essential for translational fidelity. In support, we have used phenotypic, reporter and protein-based assays to demonstrate increased translational infidelity in trm9Δ Saccharomyces cerevisiae cells. Codon reengineering studies suggest that Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific genes, those rich in arginine and glutamic acid codons from mixed boxes. Using quantitative tRNA modification analysis, we determined that trm9Δ cells are only deficient in 2 of 23 tRNA modifications, with those 2, 5-methoxycarbonylmethyluridine (mcm ( 5) U) and 5-methoxycarbonylmethyl-2-thiouridine (mcm ( 5) s ( 2) U), classified as key determinants of translational fidelity. We also show that in the absence of mcm ( 5) U and mcm ( 5) s ( 2) U, the resulting translational infidelity promotes protein errors and activation of unfolded protein and heat shock responses. These data support a model in which Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific transcripts, with decreased wobble base modification leading to translational infidelity, protein errors and activation of protein stress response pathways. PMID:22832247

  17. tRNA Modification Enzymes GidA and MnmE: Potential Role in Virulence of Bacterial Pathogens

    PubMed Central

    Shippy, Daniel C.; Fadl, Amin A.

    2014-01-01

    Transfer RNA (tRNA) is an RNA molecule that carries amino acids to the ribosomes for protein synthesis. These tRNAs function at the peptidyl (P) and aminoacyl (A) binding sites of the ribosome during translation, with each codon being recognized by a specific tRNA. Due to this specificity, tRNA modification is essential for translational efficiency. Many enzymes have been implicated in the modification of bacterial tRNAs, and these enzymes may complex with one another or interact individually with the tRNA. Approximately, 100 tRNA modification enzymes have been identified with glucose-inhibited division (GidA) protein and MnmE being two of the enzymes studied. In Escherichia coli and Salmonella, GidA and MnmE bind together to form a functional complex responsible for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm5s2U34) of tRNAs. Studies have implicated this pathway in a major pathogenic regulatory mechanism as deletion of gidA and/or mnmE has attenuated several bacterial pathogens like Salmonella enterica serovar Typhimurium, Pseudomonas syringae, Aeromonas hydrophila, and many others. In this review, we summarize the potential role of the GidA/MnmE tRNA modification pathway in bacterial virulence, interactions with the host, and potential therapeutic strategies resulting from a greater understanding of this regulatory mechanism. PMID:25310651

  18. Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis.

    PubMed

    Remus, Barbara S; Schwer, Beate; Shuman, Stewart

    2016-10-01

    Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3'-OH, 2'-PO4, and 5'-PO4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and they are promising targets for antifungal drug discovery because: (i) their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme; and (ii) there are no obvious homologs of the Trl1 ligase domain in mammalian proteomes. Here we characterize the tRNA ligases of two human fungal pathogens: Coccidioides immitis and Aspergillus fumigatus The biological activity of CimTrl1 and AfuTrl1 was verified by showing that their expression complements a Saccharomyces cerevisiae trl1Δ mutant. Purified recombinant AfuTrl1 and CimTrl1 proteins were catalytically active in joining 2',3'-cyclic-PO4 and 5'-OH ends in vitro, either as full-length proteins or as a mixture of separately produced healing and sealing domains. The biochemical properties of CimTrl1 and AfuTrl1 are similar to those of budding yeast Trl1, particularly with respect to their preferential use of GTP as the phosphate donor for the polynucleotide kinase reaction. Our findings provide genetic and biochemical tools to screen for inhibitors of tRNA ligases from pathogenic fungi.

  19. Head swivel on the ribosome facilitates translocation via intra-subunit tRNA hybrid sites

    PubMed Central

    Ratje, Andreas H.; Loerke, Justus; Mikolajka, Aleksandra; Brünner, Matthias; Hildebrand, Peter W.; Starosta, Agata L.; Dönhöfer, Alexandra; Connell, Sean R.; Fucini, Paola; Mielke, Thorsten; Whitford, Paul C.; Onuchic, Jose’ N; Yu, Yanan; Sanbonmatsu, Karissa Y.; Hartmann, Roland K.; Penczek, Pawel A.; Wilson, Daniel N.; Spahn, Christian M.T.

    2011-01-01

    The elongation cycle of protein synthesis involves the delivery of aminoacyl-tRNAs to the A-site of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A-site1,2. The translocation reaction is catalyzed by elongation factor G (EF-G) in a GTP-dependent fashion3. Despite the availability of structures of various EF-G-ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryo-EM analysis to resolve two previously unseen subpopulations within EF-G-ribosome complexes at sub-nanometer resolution, one of them with a partially translocated tRNA. Comparison of these sub-states reveals that translocation of tRNA on the 30S subunit parallels the swiveling of the 30S-head and is coupled to un-ratcheting of the 30S-body. Since the tRNA maintains contact with the P-site on the 30S-head and simultaneously establishes interaction with the E-site on the 30S-platform, a novel intra-subunit pe/E hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swiveled 30S-head conformation. These findings provide direct structural and mechanistic insight into the “missing link” in terms of tRNA intermediates involved in the universally conserved translocation process. PMID:21124459

  20. Source of amino acids for tRNA acylation. Implications for measurement of protein synthesis.

    PubMed Central

    Barnes, D M; Calvert, C C; Klasing, K C

    1992-01-01

    Estimates of protein-synthesis rates using radioisotopes require accurate measurement of the specific radioactivity of the label in protein and in the precursor pool over time. Although the extracellular and intracellular pools of amino acids are easiest to sample, the tRNA pool is the direct precursor and is the appropriate pool for sampling. To test if the intracellular or extracellular pools reflect the tRNA specific radioactivity, a chicken macrophage cell line was incubated in medium containing either 0.23 mM-leucine and 14.5 microCi of [3H]leucine (tracer dose) or 2.3 microM-leucine plus 145.0 microCi of [3H]leucine (flooding dose). At both leucine levels, the tRNA specific radioactivity reached a plateau quickly, but did not equilibrate with either the extracellular or intracellular specific radioactivity within 30 min, and remained closer to that of protein. In a second experiment, proteins in chicken macrophages were labelled with [3H]leucine for 2 days. Labelling medium was removed, and the cells were washed free of residual free [3H]leucine and incubated with medium containing either 0.23 mM- or 2.3 mM-leucine (unlabelled). The specific radioactivity of leucyl-tRNA leucine reached a plateau within 2 min and remained considerably closer to that in the protein than that in intracellular or extracellular pools for at least 60 min. These results suggest that amino acids from protein degradation are a primary source for charging tRNA. When protein-synthesis rates are estimated by label incorporation, use of extracellular or intracellular specific-radioactivity values result in a marked underestimation. PMID:1575701

  1. The core domain of Aquifex aeolicus tRNA (m7G46) methyltransferase has the methyl-transfer activity to tRNA.

    PubMed

    Tomikawa, Chie; Hori, Hiroyuki

    2006-01-01

    Transfer RNA (m(7)G46) methyltransferase [TrmB] catalyses the transfer of methyl groups from S-adenosyl-L-methionine to the N(7)-atom of guanine at position 46 in tRNA. TrmB proteins from thermophilic bacteria such as Aquifex aeolicus have a long C-terminal region as compared to those from mesophilic bacteria. Further, N-terminal region observed in TrmB proteins from mesophiles is missing in A. aeolicus TrmB. Therefore, we considered that this distinct C-terminal region in A. aeolicus TrmB might compensate the N-terminal region in mesophile TrmB and function as a part of tRNA binding site. To confirm this idea, we deleted the C-terminal region by introduction of the stop codon at position 202. To our surprise, methyl-transfer assay using yeast tRNA(Phe) transcript clearly showed that the resultant mutant protein (Glu202Stop) had an enzymatic activity. Thus, the core domain of the A. aeolicus TrmB has a methyl-transfer activity.

  2. The yeast rapid tRNA decay pathway primarily monitors the structural integrity of the acceptor and T-stems of mature tRNA

    PubMed Central

    Whipple, Joseph M.; Lane, Elizabeth A.; Chernyakov, Irina; D'Silva, Sonia; Phizicky, Eric M.

    2011-01-01

    tRNAs, like other RNAs, are subject to quality control steps during and after biosynthesis. We previously described a rapid tRNA degradation (RTD) pathway in which the 5′–3′ exonucleases Rat1 and Xrn1 degrade mature tRNAVal(AAC) in yeast mutants lacking m7G and m5C, and mature tRNASer(CGA) in mutants lacking Um and ac4C. To understand how the RTD pathway selects substrate tRNAs among different tRNAs lacking the same modifications, we used a genetic screen to examine tRNASer(CGA) variants. Our results suggest that RTD substrate recognition in vivo depends primarily on the stability of the acceptor and T-stems, and not the anti-codon stem, and does not necessarily depend on modifications, since fully modified tRNAs are subject to RTD if appropriately destabilized. We found that weaker predicted stability of the acceptor and T-stems of tRNAs is strongly correlated with RTD sensitivity, increased RNase T2 sensitivity of this region of the tRNA in vitro, and increased exposure of the 5′ end to phosphatase. We also found that purified Xrn1 selectively degrades RTD substrate tRNAs in vitro under conditions in which nonsubstrates are immune. These results suggest that tRNAs have evolved not only for accurate translation, but for resistance to attack by RTD. PMID:21632824

  3. 5 prime -Azido-(3,6- sup 3 H sub 2 )-1-naphthylphthalamic acid, a photoactivatable probe for naphthylphthalamic acid receptor proteins from higher plants: Identification of a 23-kDa protein from maize coleoptile plasma membranes

    SciTech Connect

    Zettl, R.; Feldwisch, J.; Schell, J.; Palme, K. ); Boland, W. )

    1992-01-15

    1-Naphthylphthalamic acid (NPA) is a specific inhibitor of polar auxin transport that blocks carrier mediated auxin efflux from plant cells. To allow identification of the NPA receptor thought to be part of the auxin efflux carrier, the authors have synthesized a tritiated, photolabile NPA analogue, 5{prime}-azido-(3,6-{sup 3}H{sub 2})NPA (({sup 3}H{sub 2})N{sub 3}NPA). This analogue was used to identify NPA-binding proteins in fractions highly enriched for plasma membrane vesicles isolated from maize coleoptiles (Zea mays L.). Competition studies showed that binding of ({sup 3}H{sub 2})N{sub 3}NPA to maize plasma membrane vesicles was blocked by nonradioactive NPA but not by benzoic acid. After incubation of plasma membrane vesicles with ({sup 3}H{sub 2})N{sub 3}NPA and exposure to UV light, they observed specific photoaffinity labeling of a protein with an apparent molecular mass of 23 kDa. Pretreatment of the plasma membrane vesicles with indole-3-acetic acid or with the auxin-transport inhibitors NPA and 2,3,5-triiodobenzoic acid strongly reduced specific labeling of this protein. This 23-kDa protein was also labeled by addition of 5-azido-(7-{sup 3}H)indole-3-acetic acid to plasma membranes prior to exposure to UV light. The 23-kDa protein was solubilized from plasma membranes by 1% Triton X-100. The possibility that this 23-kDa polypeptide is part of the auxin efflux carrier system is discussed.

  4. Structure-Function Analysis of Rny1 in tRNA Cleavage and Growth Inhibition

    PubMed Central

    Luhtala, Natalie; Parker, Roy

    2012-01-01

    T2 ribonucleases are conserved nucleases that affect a variety of processes in eukaryotic cells including the regulation of self-incompatibility by S-RNases in plants, modulation of host immune cell responses by viral and schistosome T2 enzymes, and neurological development and tumor progression in humans. These roles for RNaseT2’s can be due to catalytic or catalytic-independent functions of the molecule. Despite this broad importance, the features of RNaseT2 proteins that modulate catalytic and catalytic-independent functions are poorly understood. Herein, we analyze the features of Rny1 in Saccharomyces cerevisiae to determine the requirements for cleaving tRNA in vivo and for inhibiting cellular growth in a catalytic-independent manner. We demonstrate that catalytic-independent inhibition of growth is a combinatorial property of the protein and is affected by a fungal-specific C-terminal extension, the conserved catalytic core, and the presence of a signal peptide. Catalytic functions of Rny1 are independent of the C-terminal extension, are affected by many mutations in the catalytic core, and also require a signal peptide. Biochemical flotation assays reveal that in rny1Δ cells, some tRNA molecules associate with membranes suggesting that cleavage of tRNAs by Rny1 can involve either tRNA association with, or uptake into, membrane compartments. PMID:22829915

  5. Silent Polymorphisms: Can the tRNA Population Explain Changes in Protein Properties?

    PubMed Central

    Fernández-Calero, Tamara; Cabrera-Cabrera, Florencia; Ehrlich, Ricardo; Marín, Mónica

    2016-01-01

    Silent mutations are being intensively studied. We previously showed that the estrogen receptor alpha Ala87’s synonymous polymorphism affects its functional properties. Whereas a link has been clearly established between the effect of silent mutations, tRNA abundance and protein folding in prokaryotes, this connection remains controversial in eukaryotic systems. Although a synonymous polymorphism can affect mRNA structure or the interaction with specific ligands, it seems that the relative frequencies of isoacceptor tRNAs could play a key role in the protein-folding process, possibly through modulation of translation kinetics. Conformational changes could be subtle but enough to cause alterations in solubility, proteolysis profiles, functional parameters or intracellular targeting. Interestingly, recent advances describe dramatic changes in the tRNA population associated with proliferation, differentiation or response to chemical, physical or biological stress. In addition, several reports reveal changes in tRNAs’ posttranscriptional modifications in different physiological or pathological conditions. In consequence, since changes in the cell state imply quantitative and/or qualitative changes in the tRNA pool, they could increase the likelihood of protein conformational variants, related to a particular codon usage during translation, with consequences of diverse significance. These observations emphasize the importance of genetic code flexibility in the co-translational protein-folding process. PMID:26901226

  6. Unique pathway of expression of an opal suppressor phosphoserine tRNA.

    PubMed Central

    Lee, B J; de la Peña, P; Tobian, J A; Zasloff, M; Hatfield, D

    1987-01-01

    An opal suppressor phosphoserine tRNA gene is present in single copy in the genomes of higher vertebrates. We have shown that the product of this gene functions as a suppressor in an in vitro assay, and we have proposed that it may donate a modified amino acid directly to protein in response to specific UGA codons. In this report, we show through in vitro and in vivo studies that the human and Xenopus opal suppressor phosphoserine tRNAs are synthesized by a pathway that is, to the best of our knowledge, unlike that of any known eukaryotic tRNA. The primary transcript of this gene does not contain a 5'-leader sequence; and, therefore, transcription of this suppressor is initiated at the first nucleotide within the coding sequence. The 5'-terminal triphosphate, present on the primary transcript, remains intact through 3'-terminal maturation and through subsequent transport of the tRNA to the cytoplasm. The unique biosynthetic pathway of this opal suppressor may underlie its distinctive role in eukaryotic cells. Images PMID:3114749

  7. Mitochondrial genomes of praying mantises (Dictyoptera, Mantodea): rearrangement, duplication, and reassignment of tRNA genes

    PubMed Central

    Ye, Fei; Lan, Xu-e; Zhu, Wen-bo; You, Ping

    2016-01-01

    Insect mitochondrial genomes (mitogenomes) contain a conserved set of 37 genes for an extensive diversity of lineages. Previously reported dictyopteran mitogenomes share this conserved mitochondrial gene arrangement, although surprisingly little is known about the mitogenome of Mantodea. We sequenced eight mantodean mitogenomes including the first representatives of two families: Hymenopodidae and Liturgusidae. Only two of these genomes retain the typical insect gene arrangement. In three Liturgusidae species, the trnM genes have translocated. Four species of mantis (Creobroter gemmata, Mantis religiosa, Statilia sp., and Theopompa sp.-HN) have multiple identical tandem duplication of trnR, and Statilia sp. additionally includes five extra duplicate trnW. These extra trnR and trnW in Statilia sp. are erratically arranged and form another novel gene order. Interestingly, the extra trnW is converted from trnR by the process of point mutation at anticodon, which is the first case of tRNA reassignment for an insect. Furthermore, no significant differences were observed amongst mantodean mitogenomes with variable copies of tRNA according to comparative analysis of codon usage. Combined with phylogenetic analysis, the characteristics of tRNA only possess limited phylogenetic information in this research. Nevertheless, these features of gene rearrangement, duplication, and reassignment provide valuable information toward understanding mitogenome evolution in insects. PMID:27157299

  8. Spontaneous reverse movement of mRNA-bound tRNA through the ribosome.

    PubMed

    Konevega, Andrey L; Fischer, Niels; Semenkov, Yuri P; Stark, Holger; Wintermeyer, Wolfgang; Rodnina, Marina V

    2007-04-01

    During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

  9. Unique pathway of expression of an opal suppressor phosphoserine tRNA

    SciTech Connect

    Lee, B.J.; De La Pena, P.; Tobian, J.A.; Zasloff, M.; Hatfield, D.

    1987-09-01

    An opal suppressor phosphoserine tRNA gene is present in single copy in the genomes of higher vertebrates. The authors have shown that the product of this gene functions as a suppressor in an in vitro assay, and they have proposed that it may donate a modified amino acid directly to protein in response to specific UGA codons. In this report, they show through in vitro and in vivo studies that the human and Xenopus opal suppressor phosphoserine tRNAs are synthesized by a pathway that is, to the best of our knowledge, unlike that of nay know eukaryotic tRNA. The primary transcript of this gene does not contain a 5'-leader sequence; and, therefore, transcription of this suppressor is initiated at the first nucleotide within the coding sequence. The 5'-terminal triphosphate, present on the primary transcript, remains intact through 3'-terminal maturation and through subsequent transport of the tRNA to the cytoplasm. The unique biosynthetic pathway of this opal suppressor may underlie its distinctive role in eukaryotic cells.

  10. Codon usage and tRNA content in unicellular and multicellular organisms.

    PubMed

    Ikemura, T

    1985-01-01

    Choices of synonymous codons in unicellular organisms are here reviewed, and differences in synonymous codon usages between Escherichia coli and the yeast Saccharomyces cerevisiae are attributed to differences in the actual populations of isoaccepting tRNAs. There exists a strong positive correlation between codon usage and tRNA content in both organisms, and the extent of this correlation relates to the protein production levels of individual genes. Codon-choice patterns are believed to have been well conserved during the course of evolution. Examination of silent substitutions and tRNA populations in Enterobacteriaceae revealed that the evolutionary constraint imposed by tRNA content on codon usage decelerated rather than accelerated the silent-substitution rate, at least insofar as pairs of taxonomically related organisms were examined. Codon-choice patterns of multicellular organisms are briefly reviewed, and diversity in G+C percentage at the third position of codons in vertebrate genes--as well as a possible causative factor in the production of this diversity--is discussed. PMID:3916708

  11. Horizontal gene transfer of chlamydial-like tRNA genes into early vascular plant mitochondria.

    PubMed

    Knie, Nils; Polsakiewicz, Monika; Knoop, Volker

    2015-03-01

    Mitochondrial genomes of lycophytes are surprisingly diverse, including strikingly different transfer RNA (tRNA) gene complements: No mitochondrial tRNA genes are present in the spikemoss Selaginella moellendorffii, whereas 26 tRNAs are encoded in the chondrome of the clubmoss Huperzia squarrosa. Reinvestigating the latter we found that trnL(gag) and trnS(gga) had never before been identified in any other land plant mitochondrial DNA. Sensitive sequence comparisons showed these two tRNAs as well as trnN(guu) and trnS(gcu) to be very similar to their respective counterparts in chlamydial bacteria. We identified homologs of these chlamydial-type tRNAs also in other lycophyte, fern, and gymnosperm DNAs, suggesting horizontal gene transfer (HGT) into mitochondria in the early vascular plant stem lineages. These findings extend plant mitochondrial HGT to affect individual tRNA genes, to include bacterial donors, and suggest that Chlamydiae on top of their recently proposed key role in primary chloroplast establishment may also have participated in early tracheophyte genome evolution.

  12. RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway

    PubMed Central

    Adachi, Kousuke; Nobe, Yuko; Kobayashi, Junya; Hirota, Kouji; Oliveira, Douglas V.; Taoka, Masato; Isobe, Toshiaki

    2014-01-01

    Ribonuclease mitochondrial RNA processing (RNase MRP) is a multifunctional ribonucleoprotein (RNP) complex that is involved in the maturation of various types of RNA including ribosomal RNA. RNase MRP consists of a potential catalytic RNA and several protein components, all of which are required for cell viability. We show here that the temperature-sensitive mutant of rmp1, the gene for a unique protein component of RNase MRP, accumulates the dimeric tRNA precursor, pre-tRNASer-Met. To examine whether RNase MRP mediates tRNA maturation, we purified the RNase MRP holoenzyme from the fission yeast Schizosaccharomyces pombe and found that the enzyme directly and selectively cleaves pre-tRNASer-Met, suggesting that RNase MRP participates in the maturation of specific tRNA in vivo. In addition, mass spectrometry–based ribonucleoproteomic analysis demonstrated that this RNase MRP consists of one RNA molecule and 11 protein components, including a previously unknown component Rpl701. Notably, limited nucleolysis of RNase MRP generated an active catalytic core consisting of partial mrp1 RNA fragments, which constitute “Domain 1” in the secondary structure of RNase MRP, and 8 proteins. Thus, the present study provides new insight into the structure and function of RNase MRP. PMID:25401760

  13. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

    PubMed Central

    Turowski, Tomasz W.; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-01-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential “housekeeping” roles. Many tRNA genes were found to generate long, 3′-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3′-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5′-exonuclease Rat1. PMID:27206856

  14. Rearrangement of mitochondrial tRNA genes in flat bugs (Hemiptera: Aradidae)

    PubMed Central

    Song, Fan; Li, Hu; Shao, Renfu; Shi, Aimin; Bai, Xiaoshuan; Zheng, Xiaorong; Heiss, Ernst; Cai, Wanzhi

    2016-01-01

    The typical insect mitochondrial (mt) genome organization, which contains a single chromosome with 37 genes, was found in the infraorder Pentatomomorpha (suborder Heteroptera). The arrangement of mt genes in these true bugs is usually the same as the ancestral mt gene arrangement of insects. Rearrangement of transfer RNA (tRNA) genes, however, has been found in two subfamilies of flat bugs (Mezirinae and Calisiinae, family Aradidae). In this study, we sequenced the complete mt genomes of four species from three other subfamilies (Aradinae, Carventinae and Aneurinae). We found tRNA gene rearrangement in all of these four species. All of the rearranged tRNA genes are located between the mitochondrial control region and cox1, indicating this region as a hotspot for gene rearrangement in flat bugs; the rearrangement is likely caused by events of tandem duplication and random deletion of genes. Furthermore, our phylogenetic and dating analyses indicated that the swap of positions between trnQ and trnI occurred ~162 million years ago (MYA) in the most recent common ancestor of the five subfamilies of flat bugs investigated to date, whereas the swap of positions between trnC and trnW occurred later in the lineage leading to Calisiinae, and the translocation of trnC and trnY occurred later than 134 MYA in the lineage leading to Aradinae. PMID:27180804

  15. Formation of the conserved pseudouridine at position 55 in archaeal tRNA.

    PubMed

    Roovers, Martine; Hale, Caryn; Tricot, Catherine; Terns, Michael P; Terns, Rebecca M; Grosjean, Henri; Droogmans, Louis

    2006-01-01

    Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.

  16. Mitochondrial genomes of praying mantises (Dictyoptera, Mantodea): rearrangement, duplication, and reassignment of tRNA genes.

    PubMed

    Ye, Fei; Lan, Xu-E; Zhu, Wen-Bo; You, Ping

    2016-01-01

    Insect mitochondrial genomes (mitogenomes) contain a conserved set of 37 genes for an extensive diversity of lineages. Previously reported dictyopteran mitogenomes share this conserved mitochondrial gene arrangement, although surprisingly little is known about the mitogenome of Mantodea. We sequenced eight mantodean mitogenomes including the first representatives of two families: Hymenopodidae and Liturgusidae. Only two of these genomes retain the typical insect gene arrangement. In three Liturgusidae species, the trnM genes have translocated. Four species of mantis (Creobroter gemmata, Mantis religiosa, Statilia sp., and Theopompa sp.-HN) have multiple identical tandem duplication of trnR, and Statilia sp. additionally includes five extra duplicate trnW. These extra trnR and trnW in Statilia sp. are erratically arranged and form another novel gene order. Interestingly, the extra trnW is converted from trnR by the process of point mutation at anticodon, which is the first case of tRNA reassignment for an insect. Furthermore, no significant differences were observed amongst mantodean mitogenomes with variable copies of tRNA according to comparative analysis of codon usage. Combined with phylogenetic analysis, the characteristics of tRNA only possess limited phylogenetic information in this research. Nevertheless, these features of gene rearrangement, duplication, and reassignment provide valuable information toward understanding mitogenome evolution in insects. PMID:27157299

  17. MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory

    PubMed Central

    Tek, Alex; Korostelev, Andrei A.; Flores, Samuel Coulbourn

    2016-01-01

    Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion. PMID:26673695

  18. Fluorescent labeling of tRNA dihydrouridine residues: Mechanism and distribution

    PubMed Central

    Kaur, Jaskiran; Raj, Monika; Cooperman, Barry S.

    2011-01-01

    Dihydrouridine (DHU) positions within tRNAs have long been used as sites to covalently attach fluorophores, by virtue of their unique chemical reactivity toward reduction by NaBH4, their abundance within prokaryotic and eukaryotic tRNAs, and the biochemical functionality of the labeled tRNAs so produced. Interpretation of experiments employing labeled tRNAs can depend on knowing the distribution of dye among the DHU positions present in a labeled tRNA. Here we combine matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) analysis of oligonucleotide fragments and thin layer chromatography to resolve and quantify sites of DHU labeling by the fluorophores Cy3, Cy5, and proflavin in Escherichia coli tRNAPhe and E. coli tRNAArg. The MALDI-MS results led us to re-examine the precise chemistry of the reactions that result in fluorophore introduction into tRNA. We demonstrate that, in contrast to an earlier suggestion that has long been unchallenged in the literature, such introduction proceeds via a substitution reaction on tetrahydrouridine, the product of NaBH4 reduction of DHU, resulting in formation of substituted tetrahydrocytidines within tRNA. PMID:21628433

  19. Identification of Aquifex aeolicus tRNA (m2(2G26) methyltransferase gene.

    PubMed

    Takeda, Hiroshi; Hori, Hiroyuki; Endo, Yaeta

    2002-01-01

    The modifications of N2,N2-dimethylguanine (m2(2)G) are found in tRNAs and rRNAs from eukarya and archaea. In tRNAs, modification at position G26 is generated by tRNA (m2(2)G26) methyltransferase, which is encoded by the corresponding gene, trm1. This enzyme catalyzes the methyl-transfer from S-adenosyl-L-methionine to the semi-conserved residue, G26, via the intermediate modified base, m2G26. Recent genome sequencing project has been reported that the putative trm1 is encoded in the genome of Aquifex aeolicus, a hyper-thermophilic eubacterium as only one exception among eubacteria. In order to confirm whether this bacterial trm1 gene product is a real tRNA (m2(2)G26) methyltransferase or not, we expressed this protein by wheat germ in vitro cell-free translation system. Our biochemical analysis clearly showed that this gene product possessed tRNA (m2(2)G26) methyltransferase activity.

  20. Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA*

    PubMed Central

    Awai, Takako; Kimura, Satoshi; Tomikawa, Chie; Ochi, Anna; Ihsanawati; Bessho, Yoshitaka; Yokoyama, Shigeyuki; Ohno, Satoshi; Nishikawa, Kazuya; Yokogawa, Takashi; Suzuki, Tsutomu; Hori, Hiroyuki

    2009-01-01

    Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m22G26) in tRNA. In the reaction, N2-guanine at position 26 (m2G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNACys has an m22G26m2G27 or m22G26m22G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m2G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme. PMID:19491098

  1. Aquifex aeolicus tRNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes transfer of methyl groups not only to guanine 26 but also to guanine 27 in tRNA.

    PubMed

    Awai, Takako; Kimura, Satoshi; Tomikawa, Chie; Ochi, Anna; Ihsanawati; Bessho, Yoshitaka; Yokoyama, Shigeyuki; Ohno, Satoshi; Nishikawa, Kazuya; Yokogawa, Takashi; Suzuki, Tsutomu; Hori, Hiroyuki

    2009-07-31

    Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

  2. Protein Kinase A Is Part of a Mechanism That Regulates Nuclear Reimport of the Nuclear tRNA Export Receptors Los1p and Msn5p

    PubMed Central

    Pierce, Jacqueline B.; van der Merwe, George

    2014-01-01

    The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors. PMID:24297441

  3. Protein kinase A is part of a mechanism that regulates nuclear reimport of the nuclear tRNA export receptors Los1p and Msn5p.

    PubMed

    Pierce, Jacqueline B; van der Merwe, George; Mangroo, Dev

    2014-02-01

    The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.

  4. Under- and over-water halves of Gyrinidae beetle eyes harbor different corneal nanocoatings providing adaptation to the water and air environments

    NASA Astrophysics Data System (ADS)

    Blagodatski, Artem; Kryuchkov, Michail; Sergeev, Anton; Klimov, Andrey A.; Shcherbakov, Maxim R.; Enin, Gennadiy A.; Katanaev, Vladimir L.

    2014-08-01

    Whirligig beetles (Gyrinidae) inhabit water surfaces and possess unique eyes which are split into the overwater and underwater parts. In this study we analyze the micro- and nanostructure of the split eyes of two Gyrinidae beetles genera, Gyrinus and Orectochilus. We find that corneae of the overwater ommatidia are covered with maze-like nanostructures, while the corneal surface of the underwater eyes is smooth. We further show that the overwater nanostructures possess no anti-wetting, but the anti-reflective properties with the spectral preference in the range of 450-600 nm. These findings illustrate the adaptation of the corneal nanocoating of the two halves of an insect's eye to two different environments. The novel natural anti-reflective nanocoating we describe may find future technological applications.

  5. Under- and over-water halves of Gyrinidae beetle eyes harbor different corneal nanocoatings providing adaptation to the water and air environments.

    PubMed

    Blagodatski, Artem; Kryuchkov, Michail; Sergeev, Anton; Klimov, Andrey A; Shcherbakov, Maxim R; Enin, Gennadiy A; Katanaev, Vladimir L

    2014-08-08

    Whirligig beetles (Gyrinidae) inhabit water surfaces and possess unique eyes which are split into the overwater and underwater parts. In this study we analyze the micro- and nanostructure of the split eyes of two Gyrinidae beetles genera, Gyrinus and Orectochilus. We find that corneae of the overwater ommatidia are covered with maze-like nanostructures, while the corneal surface of the underwater eyes is smooth. We further show that the overwater nanostructures possess no anti-wetting, but the anti-reflective properties with the spectral preference in the range of 450-600 nm. These findings illustrate the adaptation of the corneal nanocoating of the two halves of an insect's eye to two different environments. The novel natural anti-reflective nanocoating we describe may find future technological applications.

  6. Optimization protein productivity of human interleukin-2 through codon usage, gene copy number and intracellular tRNA concentration in CHO cells.

    PubMed

    Ou, Kua-Chun; Wang, Chih-Yang; Liu, Kuan-Ting; Chen, Yi-Ling; Chen, Yi-Chen; Lai, Ming-Derg; Yen, Meng-Chi

    2014-11-14

    Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host's tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.

  7. Does the HIV-1 primer activation signal interact with tRNA3Lys during the initiation of reverse transcription?

    PubMed Central

    Goldschmidt, Valérie; Ehresmann, Chantal; Ehresmann, Bernard; Marquet, Roland

    2003-01-01

    Reverse transcription of HIV-1 RNA is primed by a tRNA3Lys molecule bound at the primer binding site (PBS). Complex intermolecular interactions were proposed between tRNA3Lys and the RNA of the HIV-1 Mal isolate. Recently, an alternative interaction was proposed between the TΨC stem of tRNA3Lys and a primer activation signal (PAS) of the Lai and Hxb2 RNAs, suggesting major structural variations in the reverse transcription complex of different HIV-1 strains. Here, we analyzed mutants of the Hxb2 RNA that prevent the interaction between the PAS and tRNA3Lys or/and a complementary sequence in the viral RNA. We compared the kinetics of reverse transcription of the wild type and mutant Hxb2 RNAs, using either tRNA3Lys or an 18mer oligoribonucleotide complementary to the PBS, which cannot interact with the PAS, as primers. We also used chemical probing to test the structure of the mutant and wild type RNAs, as well as the complex formed between the later RNA and tRNA3Lys. These experiments, together with the analysis of long term replication data of mutant viruses obtained by C. Morrow and coworkers (Birmingham, USA) that use alternate tRNAs as primers, strongly suggest that the interaction between the Hxb2 PAS and tRNA3Lys does not exist. Instead, the effects of the vRNA mutations on reverse transcription seem to be linked to incorrect folding of the mutant RNAs. PMID:12560480

  8. Dietary uptake efficiency of 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl in yellow perch and rainbow trout: Role of dietary and body lipids

    SciTech Connect

    Dabrowska, H.; Fisher, S.W.; Dabrowski, K.; Staubus, A.E.

    1999-05-01

    Dietary uptake efficiency ({alpha}) and eliminate rate constants (k{sub d}) of 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl (HCBP) were determined in two fish species, yellow perch and rainbow trout, to investigate the influence of dietary and body lipid levels on bioaccumulation. Groups of juvenile fish with significant differences in percent body lipid were fed with a low-fat(LF) or high-fat(HF) diet spiked with 5 or 50 ppb of {sup 14}C-HCBP for 32 d. Thereafter, fish were fed an uncontaminated LF or HF diet to allow for elimination of HCBP. Feeding and growth rates were quantified. There were eight fish lipid-dietary lipid-HCBP concentration exposure combinations for each species. Four fish from each exposure were collected at the beginning of the study and at 10--17-d intervals during exposure and elimination periods for lipid and {sup 14}C-HCBP analysis. The {alpha} values ranged from 74 to 95% in yellow perch and from 79 to 99% in rainbow trout. The greatest {alpha} values, of 95 to 99%, were found in fish given diets with 5 ppb HCBP. Uptake of HCBP was influenced by both dietary and body lipids and depended on the current status of both lipid pools. The elimination rate constants were in the range of 0.000 to 0.004 d{sup {minus}1} in yellow perch and 0.003 to 0.010 d{sup {minus}1} in rainbow trout. No significant differences in elimination rate constants between HF and LF fish groups were found. In fish on a constant dietary lipid regime, the k{sub d} values tended to be less in HF than in LF fish. However, in fish groups offered diets with a change in lipid regime. The k{sub d} tended to be greater. Lipid x time interactions in the HF and LF fish groups undergoing a change in lipid regime indicated that the k{sub d} values, like the {alpha} values, were influenced by both lipid pools. Changes in elimination rates due to dietary/body lipid status impacted BAFs more strongly than changes in uptake efficiencies. The BAFs were in the range of 1.11 to 2

  9. Evolution of the tRNA gene family in mitochondrial genomes of five Meretrix clams (Bivalvia, Veneridae).

    PubMed

    Wu, Xiangyun; Xiao, Shu; Li, Xiaoling; Li, Lu; Shi, Wei; Yu, Ziniu

    2014-01-01

    In contrast to the extreme conservation of nuclear-encoded tRNAs, organization of the mitochondrial (mt) tRNA gene family in invertebrates is highly dynamic and rapidly evolving. While gene duplication and loss, gene isomerism, recruitment, and rearrangements have occurred sporadically in several invertebrate lineages, little is known regarding the pattern of their evolution. Comparisons of invertebrate mt genomes at a generic level can be extremely helpful in investigating evolutionary patterns of variation, as intermediate stages of the process may be identified. Variation of mitochondrial tRNA organization among Meretrix clams provides good materials to investigate mt tRNA evolution. We characterized the complete mt genome of the lyrate Asiatic hard clam Meretrix lyrata, re-annotated tRNAs of four previously sequenced Meretrix clams, and undertook an intensive comparison of tRNA gene families in these clams. Our results 1) provide evidence that the commonly observed duplication of trnM may have occurred independently in different bivalve lineages and, based on the higher degree of trnM gene similarity, may have occurred more recently than expected; 2) suggest that "horizontal" evolution may have played an important role in tRNA gene family evolution based on frequent gene duplications and gene recruitment events; and 3) reveal the first case of isoacceptor "vertical" tRNA gene recruitment (VTGR) and present the first clear evidence that VTGR allows rapid evolution of tRNAs. We identify the trnS(-UCR) gene in Meretrix clams, previously considered missing in this lineage, and speculate that trnS(-UCR) lacking the D-arm in both M. lyrata and Meretrix lamarckii may represent the ancestral status. Phylogenetic analysis based on 13 concatenate protein-coding genes provided opportunities to detect rapidly evolved tRNA genes via VTGR and gene isomerism processes. This study suggests that evolution of the mt tRNA gene family in bivalves is more complex than previously

  10. Large gene overlaps and tRNA processing in the compact mitochondrial genome of the crustacean Armadillidium vulgare.

    PubMed

    Doublet, Vincent; Ubrig, Elodie; Alioua, Abdelmalek; Bouchon, Didier; Marcadé, Isabelle; Maréchal-Drouard, Laurence

    2015-01-01

    A faithful expression of the mitochondrial DNA is crucial for cell survival. Animal mitochondrial DNA (mtDNA) presents a highly compact gene organization. The typical 16.5 kbp animal mtDNA encodes 13 proteins, 2 rRNAs and 22 tRNAs. In the backyard pillbug Armadillidium vulgare, the rather small 13.9 kbp mtDNA encodes the same set of proteins and rRNAs as compared to animal kingdom mtDNA, but seems to harbor an incomplete set of tRNA genes. Here, we first confirm the expression of 13 tRNA genes in this mtDNA. Then we show the extensive repair of a truncated tRNA, the expression of tRNA involved in large gene overlaps and of tRNA genes partially or fully integrated within protein-coding genes in either direct or opposite orientation. Under selective pressure, overlaps between genes have been likely favored for strong genome size reduction. Our study underlines the existence of unknown biochemical mechanisms for the complete gene expression of A. vulgare mtDNA, and of co-evolutionary processes to keep overlapping genes functional in a compacted mitochondrial genome.

  11. Topological constraints are major determinants of tRNA tertiary structure and dynamics and provide basis for tertiary folding cooperativity.

    PubMed

    Mustoe, Anthony M; Brooks, Charles L; Al-Hashimi, Hashim M

    2014-10-01

    Recent studies have shown that basic steric and connectivity constraints encoded at the secondary structure level are key determinants of 3D structure and dynamics in simple two-way RNA junctions. However, the role of these topological constraints in higher order RNA junctions remains poorly understood. Here, we use a specialized coarse-grained molecular dynamics model to directly probe the thermodynamic contributions of topological constraints in defining the 3D architecture and dynamics of transfer RNA (tRNA). Topological constraints alone restrict tRNA's allowed conformational space by over an order of magnitude and strongly discriminate against formation of non-native tertiary contacts, providing a sequence independent source of folding specificity. Topological constraints also give rise to long-range correlations between the relative orientation of tRNA's helices, which in turn provides a mechanism for encoding thermodynamic cooperativity between distinct tertiary interactions. These aspects of topological constraints make it such that only several tertiary interactions are needed to confine tRNA to its native global structure and specify functionally important 3D dynamics. We further show that topological constraints are conserved across tRNA's different naturally occurring secondary structures. Taken together, our results emphasize the central role of secondary-structure-encoded topological constraints in defining RNA 3D structure, dynamics and folding.

  12. A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α.

    PubMed

    Begley, Ulrike; Sosa, Maria Soledad; Avivar-Valderas, Alvaro; Patil, Ashish; Endres, Lauren; Estrada, Yeriel; Chan, Clement T Y; Su, Dan; Dedon, Peter C; Aguirre-Ghiso, Julio A; Begley, Thomas

    2013-03-01

    Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9-like (hTRM9L/KIAA1456) mRNA is down-regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re-expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence-like G0/G1-arrest and up-regulation of the RB interacting protein LIN9. Additionally, SW620 cells re-expressing hTRM9L did not respond to hypoxia via HIF1-α-dependent induction of GLUT1. Importantly, hTRM9L-negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L-expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1-α-dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L-deficient tumours. PMID:23381944

  13. T box transcription antitermination riboswitch: Influence of nucleotide sequence and orientation on tRNA binding by the antiterminator element

    PubMed Central

    Fauzi, Hamid; Agyeman, Akwasi; Hines, Jennifer V.

    2008-01-01

    Many bacteria utilize riboswitch transcription regulation to monitor and appropriately respond to cellular levels of important metabolites or effector molecules. The T box transcription antitermination riboswitch responds to cognate uncharged tRNA by specifically stabilizing an antiterminator element in the 5′-untranslated mRNA leader region and precluding formation of a thermodynamically more stable terminator element. Stabilization occurs when the tRNA acceptor end base pairs with the first four nucleotides in the seven nucleotide bulge of the highly conserved antiterminator element. The significance of the conservation of the antiterminator bulge nucleotides that do not base pair with the tRNA is unknown, but they are required for optimal function. In vitro selection was used to determine if the isolated antiterminator bulge context alone dictates the mode in which the tRNA acceptor end binds the bulge nucleotides. No sequence conservation beyond complementarity was observed and the location was not constrained to the first four bases of the bulge. The results indicate that formation of a structure that recognizes the tRNA acceptor end in isolation is not the determinant driving force for the high phylogenetic sequence conservation observed within the antiterminator bulge. Additional factors or T box leader features more likely influenced the phylogenetic sequence conservation. PMID:19152843

  14. Large gene overlaps and tRNA processing in the compact mitochondrial genome of the crustacean Armadillidium vulgare.

    PubMed

    Doublet, Vincent; Ubrig, Elodie; Alioua, Abdelmalek; Bouchon, Didier; Marcadé, Isabelle; Maréchal-Drouard, Laurence

    2015-01-01

    A faithful expression of the mitochondrial DNA is crucial for cell survival. Animal mitochondrial DNA (mtDNA) presents a highly compact gene organization. The typical 16.5 kbp animal mtDNA encodes 13 proteins, 2 rRNAs and 22 tRNAs. In the backyard pillbug Armadillidium vulgare, the rather small 13.9 kbp mtDNA encodes the same set of proteins and rRNAs as compared to animal kingdom mtDNA, but seems to harbor an incomplete set of tRNA genes. Here, we first confirm the expression of 13 tRNA genes in this mtDNA. Then we show the extensive repair of a truncated tRNA, the expression of tRNA involved in large gene overlaps and of tRNA genes partially or fully integrated within protein-coding genes in either direct or opposite orientation. Under selective pressure, overlaps between genes have been likely favored for strong genome size reduction. Our study underlines the existence of unknown biochemical mechanisms for the complete gene expression of A. vulgare mtDNA, and of co-evolutionary processes to keep overlapping genes functional in a compacted mitochondrial genome. PMID:26361137

  15. Highly Predictive Reprogramming of tRNA Modifications Is Linked to Selective Expression of Codon-Biased Genes

    PubMed Central

    2016-01-01

    Cells respond to stress by controlling gene expression at several levels, with little known about the role of translation. Here, we demonstrate a coordinated translational stress response system involving stress-specific reprogramming of tRNA wobble modifications that leads to selective translation of codon-biased mRNAs representing different classes of critical response proteins. In budding yeast exposed to four oxidants and five alkylating agents, tRNA modification patterns accurately distinguished among chemically similar stressors, with 14 modified ribonucleosides forming the basis for a data-driven model that predicts toxicant chemistry with >80% sensitivity and specificity. tRNA modification subpatterns also distinguish SN1 from SN2 alkylating agents, with SN2-induced increases in m3C in tRNA mechanistically linked to selective translation of threonine-rich membrane proteins from genes enriched with ACC and ACT degenerate codons for threonine. These results establish tRNA modifications as predictive biomarkers of exposure and illustrate a novel regulatory mechanism for translational control of cell stress response. PMID:25772370

  16. Large gene overlaps and tRNA processing in the compact mitochondrial genome of the crustacean Armadillidium vulgare

    PubMed Central

    Doublet, Vincent; Ubrig, Elodie; Alioua, Abdelmalek; Bouchon, Didier; Marcadé, Isabelle; Maréchal-Drouard, Laurence

    2015-01-01

    A faithful expression of the mitochondrial DNA is crucial for cell survival. Animal mitochondrial DNA (mtDNA) presents a highly compact gene organization. The typical 16.5 kbp animal mtDNA encodes 13 proteins, 2 rRNAs and 22 tRNAs. In the backyard pillbug Armadillidium vulgare, the rather small 13.9 kbp mtDNA encodes the same set of proteins and rRNAs as compared to animal kingdom mtDNA, but seems to harbor an incomplete set of tRNA genes. Here, we first confirm the expression of 13 tRNA genes in this mtDNA. Then we show the extensive repair of a truncated tRNA, the expression of tRNA involved in large gene overlaps and of tRNA genes partially or fully integrated within protein-coding genes in either direct or opposite orientation. Under selective pressure, overlaps between genes have been likely favored for strong genome size reduction. Our study underlines the existence of unknown biochemical mechanisms for the complete gene expression of A. vulgare mtDNA, and of co-evolutionary processes to keep overlapping genes functional in a compacted mitochondrial genome. PMID:26361137

  17. N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network

    PubMed Central

    Tomikawa, Chie; Yokogawa, Takashi; Kanai, Tamotsu; Hori, Hiroyuki

    2010-01-01

    N7-methylguanine at position 46 (m7G46) in tRNA is produced by tRNA (m7G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m7G46 modification in tRNAPhe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m7G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m1G37, suggesting that the m7G46 positively affects their formations. Although the lack of the m7G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNAPhe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNAPhe and tRNAIle. 35S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m7G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m7G46 modification supports introduction of other modifications. PMID:19934251

  18. N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network.

    PubMed

    Tomikawa, Chie; Yokogawa, Takashi; Kanai, Tamotsu; Hori, Hiroyuki

    2010-01-01

    N(7)-methylguanine at position 46 (m(7)G46) in tRNA is produced by tRNA (m(7)G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (DeltatrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the DeltatrmB strain and the lack of the m(7)G46 modification in tRNA(Phe) were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the DeltatrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m(7)G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m(1)G37, suggesting that the m(7)G46 positively affects their formations. Although the lack of the m(7)G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA(Phe), they cause a decrease in melting temperature of class I tRNA and degradation of tRNA(Phe) and tRNA(Ile). (35)S-Met incorporation into proteins revealed that protein synthesis in DeltatrmB cells is depressed above 70 degrees C. At 80 degrees C, the DeltatrmB strain exhibits a severe growth defect. Thus, the m(7)G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m(7)G46 modification supports introduction of other modifications.

  19. Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases.

    PubMed

    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Mertens, Haydyn; Svergun, Dmitri; Brieba, Luis G; Grøtli, Morten; Torres-Larios, Alfredo

    2016-07-01

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.

  20. Acquisition of an insertion peptide for efficient aminoacylation by a halophile tRNA synthetase.

    PubMed

    Evilia, Caryn; Hou, Ya-Ming

    2006-06-01

    Enzymes of halophilic organisms contain unusual peptide motifs that are absent from their mesophilic counterparts. The functions of these halophile-specific peptides are largely unknown. Here we have identified an unusual peptide that is unique to several halophile archaeal cysteinyl-tRNA synthetases (CysRS), which catalyze attachment of cysteine to tRNA(Cys) to generate the essential cysteinyl-tRNA(Cys) required for protein synthesis. This peptide is located near the active site in the catalytic domain and is highly enriched with acidic residues. In the CysRS of the extreme halophile Halobacterium species NRC-1, deletion of the peptide reduces the catalytic efficiency of aminoacylation by a factor of 100 that largely results from a defect in kcat, rather than the Km for tRNA(Cys). In contrast, maintaining the peptide length but substituting acidic residues in the peptide with neutral or basic residues has no major deleterious effect, suggesting that the acidity of the peptide is not important for the kcat of tRNA aminoacylation. Analysis of general protein structure under physiological high salt concentrations, by circular dichroism and by fluorescence titration of tRNA binding, indicates little change due to deletion of the peptide. However, the presence of the peptide confers tolerance to lower salt levels, and fluorescence analysis in 30% sucrose reveals instability of the enzyme without the peptide. We suggest that the stability associated with the peptide can be used to promote proper enzyme conformation transitions in various stages of tRNA aminoacylation that are associated with catalysis. The acquisition of the peptide by the halophilic CysRS suggests an enzyme adaptation to high salinity.

  1. Delta sequences in the 5' non-coding region of yeast tRNA genes

    PubMed Central

    Gafner, Jürg; Robertis, Eddy M.De; Philippsen, Peter

    1983-01-01

    Two so far undetected tRNA genes were found close to delta (δ) sequences at the sup4 locus on chromosome X in the genome of Saccharomyces cerevisiae. The two genes were identified from their abundant transcription products in frog oocytes. Hybridisation experiments allowed the mapping of the transcripts in cloned DNA and DNA sequence analysis revealed the presence of one AGGtRNAArg and one GACtRNAAsp gene. tRNAAsp genes with sequences similar or identical to GACtRNAAsp exist in 14-16 copies per haploid yeast genome, whereas only one copy was detected for AGGtRNAArg. In vivo labelling of total yeast tRNA with 32P followed by hybridisation revealed that the unique AGGtRNAArg gene is transcribed in S. cerevisiae. δ sequences are present 120 bp upstream from the first coding nucleotide in the case of AGGtRNAArg, 80 bp in the case of GACtRNAAsp and 405 bp in the case of the known UACtRNATyr (sup4) gene. δ sequences, as part of Ty elements or alone, were also found by other investigators at similar distances upstream of the mRNA start in mutant alleles of protein-coding yeast genes. Although protein-coding genes are transcribed by RNA polymerase II and tRNA genes by RNA polymerase III, the 5' non-coding region of both types of genes could conceivably have a peculiar DNA or chromatin structure used as preferred landing sites by transposable elements. ImagesFig. 1.Fig. 2.Fig. 5.Fig. 6. PMID:16453444

  2. Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases*♦

    PubMed Central

    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Brieba, Luis G.; Grøtli, Morten

    2016-01-01

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor. PMID:27226617

  3. RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification.

    PubMed

    Arimbasseri, Aneeshkumar G; Blewett, Nathan H; Iben, James R; Lamichhane, Tek N; Cherkasova, Vera; Hafner, Markus; Maraia, Richard J

    2015-12-01

    Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m(2)2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m(2)2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(2)2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m(2)2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(2)2G26 modification and that this response is conserved among highly divergent yeasts and human cells. PMID:26720005

  4. Was there a universal tRNA before specialized tRNAs came into existence?

    NASA Technical Reports Server (NTRS)

    Lacey, James C., Jr.; Staves, Mark P.

    1990-01-01

    It is generally true that evolving systems begin simply and become more complex in the evolutionary process. For those who try to understand the origin of a biochemical system, what is required is the development of an idea as to what simpler system preceded the present one. A hypothesis is presented that a universal tRNA molecule, capable of reading many codons, may have preceded the appearance of individual tRNAs. Evidence seems to suggest that this molecule may have been derived from a common ancestor of the contemporary 5S rRNAs and tRNAs.

  5. The nucleotide sequence of glutamate tRNA4 of Drosophila melanogaster.

    PubMed Central

    Altwegg, M; Kubli, E

    1980-01-01

    The nucleotide sequence of Drosophila melanogaster glutamate tRNA4 was determined to be: pU-C-C-C-A-U-A-U-G-G-U-C-psi-A-G-D-G-G-C-D-A-G-G-A-U-A-U-C-U-G-G-C (m) -U-U-U-C-A-C-C-A-G-A-A-G-G-C-C-C-G-G-G-T-psi-U-C-G-A-U-U-C-C-C-G-G-U-A-U-G-G-G-A-A-C-C-AOH. A partial modified C is found at position 32 in the anticodon loop. Images PMID:6775307

  6. RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification

    PubMed Central

    Arimbasseri, Aneeshkumar G.; Blewett, Nathan H.; Iben, James R.; Lamichhane, Tek N.; Cherkasova, Vera; Hafner, Markus; Maraia, Richard J.

    2015-01-01

    Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m2 2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m2 2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m2 2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m2 2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m2 2G26 modification and that this response is conserved among highly divergent yeasts and human cells. PMID:26720005

  7. Optical Kerr effect of tRNA solution induced by femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Kucia, Weronika E.; Sharma, Gargi; Joseph, Cecil S.; Sarbak, Szymon; Oliver, Cameron; Dobek, Andrzej; Giles, Robert H.

    2016-10-01

    The optical Kerr effect (OKE) in a transfer ribonucleic acid (tRNA) solution induced by femtosecond pulses of linearly polarized pump light (λi = 800 nm) and sounded by probe light (λp = 800 nm) was studied. The measurements were performed to find nonlinear optical parameters describing a single molecule (molecular Kerr constant K, mean nonlinear third order optical polarizability cpi) and to compare them with our previous OKE results obtained in ns and ps time range. The OKE experiment has proven to be an efficient method to obtain the nonlinear parameters of single molecules in solution, which reflects dynamic structure changes.

  8. Anticodon Modifications in the tRNA Set of LUCA and the Fundamental Regularity in the Standard Genetic Code.

    PubMed

    van der Gulik, Peter T S; Hoff, Wouter D

    2016-01-01

    Based on (i) an analysis of the regularities in the standard genetic code and (ii) comparative genomics of the anticodon modification machinery in the three branches of life, we derive the tRNA set and its anticodon modifications as it was present in LUCA. Previously we proposed that an early ancestor of LUCA contained a set of 23 tRNAs with unmodified anticodons that was capable of translating all 20 amino acids while reading 55 of the 61 sense codons of the standard genetic code (SGC). Here we use biochemical and genomic evidence to derive that LUCA contained a set of 44 or 45 tRNAs containing 2 or 3 modifications while reading 59 or 60 of the 61 sense codons. Subsequent tRNA modifications occurred independently in the Bacteria and Eucarya, while the Archaea have remained quite close to the tRNA set as it was present in LUCA. PMID:27454314

  9. Anticodon Modifications in the tRNA Set of LUCA and the Fundamental Regularity in the Standard Genetic Code

    PubMed Central

    van der Gulik, Peter T. S.; Hoff, Wouter D.

    2016-01-01

    Based on (i) an analysis of the regularities in the standard genetic code and (ii) comparative genomics of the anticodon modification machinery in the three branches of life, we derive the tRNA set and its anticodon modifications as it was present in LUCA. Previously we proposed that an early ancestor of LUCA contained a set of 23 tRNAs with unmodified anticodons that was capable of translating all 20 amino acids while reading 55 of the 61 sense codons of the standard genetic code (SGC). Here we use biochemical and genomic evidence to derive that LUCA contained a set of 44 or 45 tRNAs containing 2 or 3 modifications while reading 59 or 60 of the 61 sense codons. Subsequent tRNA modifications occurred independently in the Bacteria and Eucarya, while the Archaea have remained quite close to the tRNA set as it was present in LUCA. PMID:27454314

  10. The nucleotide sequences of several tRNA genes from rat mitochondria: common features and relatedness to homologous species.

    PubMed Central

    Cantatore, P; De Benedetto, C; Gadaleta, G; Gallerani, R; Kroon, A M; Holtrop, M; Lanave, C; Pepe, G; Quagliariello, C; Saccone, C; Sbisa, E

    1982-01-01

    We have determined the nucleotide sequences of thirteen rat mt tRNA genes. The features of the primary and secondary structures of these tRNAs show that those for Gln, Ser, and f-Met resemble, while those for Lys, Cys, and Trp depart strikingly from the universal type. The remainder are slightly abnormal. Among many mammalian mt DNA sequences, those of mt tRNA genes are highly conserved, thus suggesting for those genes an additional, perhaps regulatory, function. A simple evolutionary relationship between the tRNAs of animal mitochondria and those of eukaryotic cytoplasm, of lower eukaryotic mitochondria or of prokaryotes, is not evident owing to the extreme divergence of the tRNA sequences in the two groups. However, a slightly higher homology does exist between a few animal mt tRNAs and those from prokaryotes or from lower eukaryotic mitochondria. PMID:7099963

  11. Accurate energies of hydrogen bonded nucleic acid base pairs and triplets in tRNA tertiary interactions

    PubMed Central

    2006-01-01

    Tertiary interactions are crucial in maintaining the tRNA structure and functionality. We used a combined sequence analysis and quantum mechanics approach to calculate accurate energies of the most frequent tRNA tertiary base pairing interactions. Our analysis indicates that six out of the nine classical tertiary interactions are held in place mainly by H-bonds between the bases. In the remaining three cases other effects have to be considered. Tertiary base pairing interaction energies range from −8 to −38 kcal/mol in yeast tRNAPhe and are estimated to contribute roughly 25% of the overall tRNA base pairing interaction energy. Six analyzed posttranslational chemical modifications were shown to have minor effect on the geometry of the tertiary interactions. Modifications that introduce a positive charge strongly stabilize the corresponding tertiary interactions. Non-additive effects contribute to the stability of base triplets. PMID:16461956

  12. tRNA 3′ processing in yeast involves tRNase Z, Rex1, and Rrp6

    PubMed Central

    Skowronek, Ewa; Grzechnik, Pawel; Späth, Bettina; Marchfelder, Anita; Kufel, Joanna

    2014-01-01

    Mature tRNA 3′ ends in the yeast Saccharomyces cerevisiae are generated by two pathways: endonucleolytic and exonucleolytic. Although two exonucleases, Rex1 and Rrp6, have been shown to be responsible for the exonucleolytic trimming, the identity of the endonuclease has been inferred from other systems but not confirmed in vivo. Here, we show that the yeast tRNA 3′ endonuclease tRNase Z, Trz1, is catalyzing endonucleolytic tRNA 3′ processing. The majority of analyzed tRNAs utilize both pathways, with a preference for the endonucleolytic one. However, 3′-end processing of precursors with long 3′ trailers depends to a greater extent on Trz1. In addition to its function in the nucleus, Trz1 processes the 3′ ends of mitochondrial tRNAs, contributing to the general RNA metabolism in this organelle. PMID:24249226

  13. tRNA acceptor stem and anticodon bases form independent codes related to protein folding.

    PubMed

    Carter, Charles W; Wolfenden, Richard

    2015-06-16

    Aminoacyl-tRNA synthetases recognize tRNA anticodon and 3' acceptor stem bases. Synthetase Urzymes acylate cognate tRNAs even without anticodon-binding domains, in keeping with the possibility that acceptor stem recognition preceded anticodon recognition. Representing tRNA identity elements with two bits per base, we show that the anticodon encodes the hydrophobicity of each amino acid side-chain as represented by its water-to-cyclohexane distribution coefficient, and this relationship holds true over the entire temperature range of liquid water. The acceptor stem codes preferentially for the surface area or size of each side-chain, as represented by its vapor-to-cyclohexane distribution coefficient. These orthogonal experimental properties are both necessary to account satisfactorily for the exposed surface area of amino acids in folded proteins. Moreover, the acceptor stem codes correctly for β-branched and carboxylic acid side-chains, whereas the anticodon codes for a wider range of such properties, but not for size or β-branching. These and other results suggest that genetic coding of 3D protein structures evolved in distinct stages, based initially on the size of the amino acid and later on its compatibility with globular folding in water.

  14. tRNA acceptor stem and anticodon bases form independent codes related to protein folding

    PubMed Central

    Carter, Charles W.; Wolfenden, Richard

    2015-01-01

    Aminoacyl-tRNA synthetases recognize tRNA anticodon and 3′ acceptor stem bases. Synthetase Urzymes acylate cognate tRNAs even without anticodon-binding domains, in keeping with the possibility that acceptor stem recognition preceded anticodon recognition. Representing tRNA identity elements with two bits per base, we show that the anticodon encodes the hydrophobicity of each amino acid side-chain as represented by its water-to-cyclohexane distribution coefficient, and this relationship holds true over the entire temperature range of liquid water. The acceptor stem codes preferentially for the surface area or size of each side-chain, as represented by its vapor-to-cyclohexane distribution coefficient. These orthogonal experimental properties are both necessary to account satisfactorily for the exposed surface area of amino acids in folded proteins. Moreover, the acceptor stem codes correctly for β-branched and carboxylic acid side-chains, whereas the anticodon codes for a wider range of such properties, but not for size or β-branching. These and other results suggest that genetic coding of 3D protein structures evolved in distinct stages, based initially on the size of the amino acid and later on its compatibility with globular folding in water. PMID:26034281

  15. Protozoan ALKBH8 oxygenases display both DNA repair and tRNA modification activities.

    PubMed

    Zdżalik, Daria; Vågbø, Cathrine B; Kirpekar, Finn; Davydova, Erna; Puścian, Alicja; Maciejewska, Agnieszka M; Krokan, Hans E; Klungland, Arne; Tudek, Barbara; van den Born, Erwin; Falnes, Pål Ø

    2014-01-01

    The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins.

  16. Two-subunit enzymes involved in eukaryotic post-transcriptional tRNA modification

    PubMed Central

    Guy, Michael P; Phizicky, Eric M

    2014-01-01

    tRNA modifications are crucial for efficient and accurate protein translation, with defects often linked to disease. There are 7 cytoplasmic tRNA modifications in the yeast Saccharomyces cerevisiae that are formed by an enzyme consisting of a catalytic subunit and an auxiliary protein, 5 of which require only a single subunit in bacteria, and 2 of which are not found in bacteria. These enzymes include the deaminase Tad2-Tad3, and the methyltransferases Trm6-Trm61, Trm8-Trm82, Trm7-Trm732, and Trm7-Trm734, Trm9-Trm112, and Trm11-Trm112. We describe the occurrence and biological role of each modification, evidence for a required partner protein in S. cerevisiae and other eukaryotes, evidence for a single subunit in bacteria, and evidence for the role of the non-catalytic binding partner. Although it is unclear why these eukaryotic enzymes require partner proteins, studies of some 2-subunit modification enzymes suggest that the partner proteins help expand substrate range or allow integration of cellular activities. PMID:25625329

  17. Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi.

    PubMed

    Glatt, Sebastian; Zabel, Rene; Kolaj-Robin, Olga; Onuma, Osita F; Baudin, Florence; Graziadei, Andrea; Taverniti, Valerio; Lin, Ting-Yu; Baymann, Frauke; Séraphin, Bertrand; Breunig, Karin D; Müller, Christoph W

    2016-09-01

    During translation elongation, decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons are receiving increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs. Mutations in Elongator subunits are associated with certain neurodegenerative diseases and cancer. Here we present the crystal structure of D. mccartyi Elp3 (DmcElp3) at 2.15-Å resolution. Our results reveal an unexpected arrangement of Elp3 lysine acetyltransferase (KAT) and radical S-adenosyl methionine (SAM) domains, which share a large interface and form a composite active site and tRNA-binding pocket, with an iron-sulfur cluster located in the dimerization interface of two DmcElp3 molecules. Structure-guided mutagenesis studies of yeast Elp3 confirmed the relevance of our findings for eukaryotic Elp3s and should aid in understanding the cellular functions and pathophysiological roles of Elongator.

  18. Trm9-Catalyzed tRNA Modifications Regulate Global Protein Expression by Codon-Biased Translation

    PubMed Central

    Deng, Wenjun; Babu, I. Ramesh; Su, Dan; Yin, Shanye; Begley, Thomas J.; Dedon, Peter C.

    2015-01-01

    Post-transcriptional modifications of transfer RNAs (tRNAs) have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5) and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modifications catalyzed by tRNA methyltransferase 9 (Trm9) in tRNAArg(UCU) and tRNAGlu(UUC) and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell. PMID:26670883

  19. From End to End: tRNA Editing at 5'- and 3'-Terminal Positions

    PubMed Central

    Betat, Heike; Long, Yicheng; Jackman, Jane E.; Mörl, Mario

    2014-01-01

    During maturation, tRNA molecules undergo a series of individual processing steps, ranging from exo- and endonucleolytic trimming reactions at their 5'- and 3'-ends, specific base modifications and intron removal to the addition of the conserved 3'-terminal CCA sequence. Especially in mitochondria, this plethora of processing steps is completed by various editing events, where base identities at internal positions are changed and/or nucleotides at 5'- and 3'-ends are replaced or incorporated. In this review, we will focus predominantly on the latter reactions, where a growing number of cases indicate that these editing events represent a rather frequent and widespread phenomenon. While the mechanistic basis for 5'- and 3'-end editing differs dramatically, both reactions represent an absolute requirement for generating a functional tRNA. Current in vivo and in vitro model systems support a scenario in which these highly specific maturation reactions might have evolved out of ancient promiscuous RNA polymerization or quality control systems. PMID:25535083

  20. Meta-regulation of Arabidopsis auxin responses depends on tRNA maturation.

    PubMed

    Leitner, Johannes; Retzer, Katarzyna; Malenica, Nenad; Bartkeviciute, Rasa; Lucyshyn, Doris; Jäger, Gunilla; Korbei, Barbara; Byström, Anders; Luschnig, Christian

    2015-04-28

    Polar transport of the phytohormone auxin throughout plants shapes morphogenesis and is subject to stringent and specific control. Here, we identify basic cellular activities connected to translational control of gene expression as sufficient to specify auxin-mediated development. Mutants in subunits of Arabidopsis Elongator, a protein complex modulating translational efficiency via maturation of tRNAs, exhibit defects in auxin-controlled developmental processes, associated with reduced abundance of PIN-formed (PIN) auxin transport proteins. Similar anomalies are observed upon interference with tRNA splicing by downregulation of RNA ligase (AtRNL), pointing to a general role of tRNA maturation in auxin signaling. Elongator Protein 6 (ELP6) and AtRNL expression patterns underline an involvement in adjusting PIN protein levels, whereas rescue of mutant defects by auxin indicates rate-limiting activities in auxin-controlled organogenesis. This emphasizes mechanisms in which auxin serves as a bottleneck for plant morphogenesis, translating common cellular activities into defined developmental readouts.

  1. Structural basis for substrate binding, cleavage and allostery in the tRNA maturase RNase Z.

    PubMed

    Li de la Sierra-Gallay, Inés; Pellegrini, Olivier; Condon, Ciarán

    2005-02-10

    Transfer RNAs (tRNAs) are synthesized as part of longer primary transcripts that require processing of both their 3' and 5' extremities in every living organism known. The 5' side is processed (matured) by the ubiquitously conserved endonucleolytic ribozyme, RNase P, whereas removal of the 3' tails can be either exonucleolytic or endonucleolytic. The endonucleolytic pathway is catalysed by an enzyme known as RNase Z, or 3' tRNase. RNase Z cleaves precursor tRNAs immediately after the discriminator base (the unpaired nucleotide 3' to the last base pair of the acceptor stem, used as an identity determinant by many aminoacyl-tRNA synthetases) in most cases, yielding a tRNA primed for addition of the CCA motif by nucleotidyl transferase. Here we report the crystal structure of Bacillus subtilis RNase Z at 2.1 A resolution, and propose a mechanism for tRNA recognition and cleavage. The structure explains the allosteric properties of the enzyme, and also sheds light on the mechanisms of inhibition by the CCA motif and long 5' extensions. Finally, it highlights the extraordinary adaptability of the metallo-hydrolase domain of the beta-lactamase family for the hydrolysis of covalent bonds. PMID:15654328

  2. Structure of tRNA Dimethylallyltransferase: RNA Modification through a Channel

    SciTech Connect

    Xie, Wei; Zhou, Chun; Huang, Raven H.

    2008-09-04

    Dimethylallyltransferase (DMATase) transfers a five-carbon isoprenoid moiety from dimethylallyl pyrophosphate (DMAPP) to the amino group of adenosine at position 37 of certain tRNAs. Reported here are the crystal structures of Pseudomonas aeruginosa DMATase alone and in complex with pyrophosphate at 1.9 {angstrom} resolution. Surprisingly, the enzyme possesses a central channel spanning the entire width of the enzyme. Both the accepting substrate tRNA and the donating substrate DMAPP appear to enter the channel from opposite sides in an ordered sequence, with tRNA first and DMAPP second, and the RNA modification reaction occurs in the middle of the channel once the two substrates have met. The structure of DMATase is homologous to a class of small soluble kinases involved in biosynthesis of nucleotide precursors for nucleic acids, indicating its possibly evolutionary origin. Furthermore, specific recognition of the pyrophosphate by a conserved loop in DMATase, similar to the P-loop commonly seen in diverse nucleotide-binding proteins, demonstrates that DMATase is structurally and mechanistically distinct from farnesyltransferase, another family of prenyltransferases involved in protein modification.

  3. Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities

    PubMed Central

    Zdżalik, Daria; Vågbø, Cathrine B.; Kirpekar, Finn; Davydova, Erna; Puścian, Alicja; Maciejewska, Agnieszka M.; Krokan, Hans E.; Klungland, Arne; Tudek, Barbara; van den Born, Erwin; Falnes, Pål Ø.

    2014-01-01

    The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1–8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins. PMID:24914785

  4. Changes in tRNA methyltransferase activity and cellular S-adenosylmethionine content following methionine deprivation.

    PubMed

    Tisdale, M J

    1980-09-19

    Although homocysteine was unable to support growth of Walker carcinoma in media lacking methionine it did enable some proliferation of TLX5 lymphoma. In both cell lines there was an increase in growth rate in the presence of homocysteine at limiting methionine concentrations. The proliferation rate of Walker carcinoma was proportional to the methionine concentraion of the medium down to 0.5 microgram/ml, whereas growth of TLX5 lymphoma was only slightly reduced at such methionine concentrations. The difference in proliferative ability between the two cell lines was reflected in the level of S-adenosyl-L-methionine under conditions of methionine deprivation. In both cases transferance to a media in which methionine was growth limiting caused a rapid increase in the activity of tRNA methyltransferases to levels six to seven-fold greater than the control. The initial increase in methylase activity was not prevented by cycloheximide, although after 4 h there was a progressive decrease in activity which approached control values within 24 h. The increase in tRNA methyltransferase activity on removal of the normal level of methionine in the medium was also seen with human embryonic fibroblasts, which are able to proliferate normally in methionine-deficient, homocysteine-supplemented media. These results suggest that methyltransferase activity may be regulated in part by the S-adenosyl-methionine content of the cell.

  5. Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry

    PubMed Central

    Mengel-Jørgensen, Jonas; Kirpekar, Finn

    2002-01-01

    Mass spectrometry plays a central role in the characterisation of modified nucleotides, but pseudouridine is a mass-silent post-transcriptional modification and hence not detectable by direct mass spectrometric analysis. We show by the use of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry that pseudouridines in tRNA can be specifically cyanoethylated by acrylonitrile without affecting the uridines. The tRNA was cyanoethylated and then subjected to digestion with either RNase A or RNase T1. Cyanoethylated digestion fragments were identified by mass spectrometric comparison of untreated and acrylonitrile-treated samples, where the addition of one acrylonitrile resulted in a mass increment of 53.0 Da. The exact modified nucleotide could be identified by tandem mass spectrometry on the cyanoethylated digestion fragment. The methodology was used to identify additional one 4-thiouridine and one pseudouridine in tRNATyrII from Escherichia coli. Furthermore, we observed that RNase A is highly tolerant towards nucleotide modifications, only being inhibited by 2′-O-methylation, whereas RNase T1 cleavage is affected by most nucleotide modifications. PMID:12466567

  6. A portable system for on-site quantification of formaldehyde in air based on G-quadruplex halves coupled with A smartphone reader.

    PubMed

    Yang, Xin; Wang, Yanru; Liu, Wei; Zhang, Yuhuan; Zheng, Fangqing; Wang, Shuaixing; Zhang, Daohong; Wang, Jianlong

    2016-01-15

    A portable colorimetric determination system based on G-quadruplex DNAzyme integrated with a smartphone was developed to quantitatively detect formaldehyde (FA). The method is based on the oxidation of FA by H2O2, which prevents the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)–H2O2 reaction catalyzed by G-quadruplex halves. With the addition of FA, the amount of the blue-green-colored free-radical cation (ABTS+) was reduced. The concentration of FA can be determined by monitoring this competitive reaction with a UV-vis spectrometer. Response surface methodology (RSM) and Box-Behnken design (BBD) were applied for optimization of the colorimetric assay. A smartphone-based colorimetric reader was also developed, which could display FA responses and report the concentration in real-time. The system could detect FA as low as 0.01 µM with a linear range of 1-600 µM. Taking advantages of smartphone and DNAzyme, the assay provides great potential for its practical application as a home testing or on-site analysis with high sensitivity and selectivity.

  7. Mitochondrial 3' tRNA editing in the jakobid Seculamonas ecuadoriensis: a novel mechanism and implications for tRNA processing.

    PubMed

    Leigh, Jessica; Lang, B Franz

    2004-04-01

    The jakobid flagellates are bacteriovorus protists with mitochondrial genomes that are the most ancestral identified to date, in that they most resemble the genomes of the alpha-proteobacterial ancestors of the mitochondrion. Because of the bacterial character of jakobid mitochondrial genomes, it was expected that mechanisms for gene expression and RNA structures would be bacterial in nature. However, sequencing of the mitochondrial genome of the jakobid Seculamonas ecuadoriensis revealed several apparent mismatches in the acceptor stems of two predicted tRNAs. To investigate this observation, we determined the cDNA sequences of these tRNAs by RT-PCR. Our results show that the last three positions of the 3' extremity, plus the discriminator position of seryl and glutamyl tRNAs, are altered posttranscriptionally, restoring orthodox base-pairing and replacing the discriminator with an adenosine residue, in an editing process that resembles that of the metazoan Lithobius forficatus. However, the most 5' of the edited nucleotides is occasionally left unedited, indicating that the editing mechanism proceeds initially by exonucleolytic degradation, followed by repair of the degraded region. This 3' tRNA editing mechanism is likely distinct from that of L. forficatus, despite the apparent similarities between the two systems.

  8. Bases in 16S rRNA important for subunit association, tRNA binding, and translocation.

    PubMed

    Shi, Xinying; Chiu, Katie; Ghosh, Srikanta; Joseph, Simpson

    2009-07-28

    Ribosomes are the cellular machinery responsible for protein synthesis. A well-orchestrated step in the elongation cycle of protein synthesis is the precise translocation of the tRNA-mRNA complex within the ribosome. Here we report the application of a new in vitro modification-interference method for the identification of bases in 16S rRNA that are essential for translocation. Our results suggest that conserved bases U56, U723, A1306, A1319, and A1468 in 16S rRNA are important for translocation. These five bases were deleted or mutated so their role in translation could be studied. Depending on the type of mutation, we observed inhibition of growth rate, subunit association, tRNA binding, and/or translocation. Interestingly, deletion of U56 or A1319 or mutation of A1319 to C showed a lethal phenotype and were defective in protein synthesis in vitro. Further analysis showed that deletion of U56 or A1319 caused defects in 30S subunit assembly, subunit association, and tRNA binding. In contrast, the A1319C mutation showed no defects in subunit association; however, the extent of tRNA binding and translocation was significantly reduced. These results show that conserved bases located as far as 100 A from the tRNA binding sites can be important for translation.

  9. A miRNA-tRNA mix-up: tRNA origin of proposed miRNA.

    PubMed

    Schopman, Nick C T; Heynen, Stephan; Haasnoot, Joost; Berkhout, Ben

    2010-01-01

    The rapid release of new data from DNA genome sequencing projects has led to a variety of misannotations in public databases. Our results suggest that next generation sequencing approaches are particularly prone to such misannotations. Two related miRNA candidates did recently enter the miRBase database, miR-1274b and miR-1274a, but they share identical 18-nucleotide stretches with tRNA (Lys3) and tRNA (Lys5) , respectively. The possibility that the small RNA fragments that led to the description of these two miRNAs originated from the two tRNAs was examined. The ratio of the miR-1274b:miR-1274a fragments does closely resemble the known tRNA lys3:lys5 ratio in the cell. Furthermore, the proposed miRNA hairpins have a very low prediction score and the proposed miRNA genes are in fact endogenous retroviral elements. We searched for other miRNA-mimics in the human genome and found more examples of tRNA-miRNA mimicry. We propose that the corresponding miRNAs should be validated in more detail, as the small RNA fragments that led to their description are likely derived from tRNA processing. PMID:20818168

  10. Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA.

    PubMed

    Honda, Shozo; Shigematsu, Megumi; Morichika, Keisuke; Telonis, Aristeidis G; Kirino, Yohei

    2015-01-01

    Transfer RNAs (tRNAs) play a central role in translation and also recently appear to have a variety of other functions in biological processes beyond translation. Here we report the development of Four-Leaf clover qRT-PCR (FL-PCR), a convenient PCR-based method, which can specifically quantify individual mature tRNA species. In FL-PCR, T4 RNA ligase 2 specifically ligates a stem-loop adapter to mature tRNAs but not to precursor tRNAs or tRNA fragments. Subsequent TaqMan qRT-PCR amplifies only unmodified regions of the tRNA-adapter ligation products; therefore, FL-PCR quantification is not influenced by tRNA post-transcriptional modifications. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types, and thus provides a much-needed simple method for analyzing tRNA abundance and heterogeneity.

  11. Cocrystal Structures of Glycyl-tRNA Synthetase in Complex with tRNA Suggest Multiple Conformational States in Glycylation*

    PubMed Central

    Qin, Xiangjing; Hao, Zhitai; Tian, Qingnan; Zhang, Zhemin; Zhou, Chun; Xie, Wei

    2014-01-01

    Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charges tRNA molecules with cognate amino acids for protein synthesis. Glycyl-tRNA synthetase (GlyRS) is one of the most intriguing aminoacyl-tRNA synthetases due to its divergent quaternary structure and abnormal charging properties. In the past decade, mutations of human GlyRS (hGlyRS) were also found to be associated with Charcot-Marie-Tooth disease. However, the mechanisms of traditional and alternative functions of hGlyRS are poorly understood due to a lack of studies at the molecular basis. In this study we report crystal structures of wild type and mutant hGlyRS in complex with tRNA and with small substrates and describe the molecular details of enzymatic recognition of the key tRNA identity elements in the acceptor stem and the anticodon loop. The cocrystal structures suggest that insertions 1 and 3 work together with the active site in a cooperative manner to facilitate efficient substrate binding. Both the enzyme and tRNA molecules undergo significant conformational changes during glycylation. A working model of multiple conformations for hGlyRS catalysis is proposed based on the crystallographic and biochemical studies. This study provides insights into the catalytic pathway of hGlyRS and may also contribute to our understanding of Charcot-Marie-Tooth disease. PMID:24898252

  12. Cocrystal structures of glycyl-tRNA synthetase in complex with tRNA suggest multiple conformational states in glycylation.

    PubMed

    Qin, Xiangjing; Hao, Zhitai; Tian, Qingnan; Zhang, Zhemin; Zhou, Chun; Xie, Wei

    2014-07-18

    Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charges tRNA molecules with cognate amino acids for protein synthesis. Glycyl-tRNA synthetase (GlyRS) is one of the most intriguing aminoacyl-tRNA synthetases due to its divergent quaternary structure and abnormal charging properties. In the past decade, mutations of human GlyRS (hGlyRS) were also found to be associated with Charcot-Marie-Tooth disease. However, the mechanisms of traditional and alternative functions of hGlyRS are poorly understood due to a lack of studies at the molecular basis. In this study we report crystal structures of wild type and mutant hGlyRS in complex with tRNA and with small substrates and describe the molecular details of enzymatic recognition of the key tRNA identity elements in the acceptor stem and the anticodon loop. The cocrystal structures suggest that insertions 1 and 3 work together with the active site in a cooperative manner to facilitate efficient substrate binding. Both the enzyme and tRNA molecules undergo significant conformational changes during glycylation. A working model of multiple conformations for hGlyRS catalysis is proposed based on the crystallographic and biochemical studies. This study provides insights into the catalytic pathway of hGlyRS and may also contribute to our understanding of Charcot-Marie-Tooth disease.

  13. The crystal structure of yeast mitochondrial ThrRS in complex with the canonical threonine tRNA

    PubMed Central

    Holman, Kaitlyn M.; Wu, Jiang; Ling, Jiqiang; Simonović, Miljan

    2016-01-01

    In mitochondria of Saccharomyces cerevisiae, a single aminoacyl-tRNA synthetase (aaRS), MST1, aminoacylates two isoacceptor tRNAs, tRNA1Thr and tRNA2Thr, that harbor anticodon loops of different size and sequence. As a result of this promiscuity, reassignment of the CUN codon box from leucine to threonine is facilitated. However, the mechanism by which a single aaRS binds distinct anticodon loops with high specificity is not well understood. Herein, we present the crystal structure of MST1 in complex with the canonical tRNA2Thr and non-hydrolyzable analog of threonyl adenylate. Our structure reveals that the dimeric arrangement of MST1 is essential for binding the 5′-phosphate, the second base pair of the acceptor stem, the first two base pairs of the anticodon stem and the first nucleotide of the variable arm. Further, in contrast to the bacterial ortholog that ‘reads’ the entire anticodon sequence, MST1 recognizes bases in the second and third position and the nucleotide upstream of the anticodon sequence. We speculate that a flexible loop linking strands β4 and β5 may be allosteric regulator that establishes cross-subunit communication between the aminoacylation and tRNA-binding sites. We also propose that structural features of the anticodon-binding domain in MST1 permit binding of the enlarged anticodon loop of tRNA1Thr. PMID:26704982

  14. Multiple Conserved Heteroplasmic Sites in tRNA Genes in the Mitochondrial Genomes of Terrestrial Isopods (Oniscidea)

    PubMed Central

    Chandler, Christopher H.; Badawi, Myriam; Moumen, Bouziane; Grève, Pierre; Cordaux, Richard

    2015-01-01

    Mitochondrial genome structure and organization are relatively conserved among metazoans. However, in many isopods, especially the terrestrial isopods (Oniscidea), the mitochondrial genome consists of both ∼14-kb linear monomers and ∼28-kb circular dimers. This unusual organization is associated with an ancient and conserved constitutive heteroplasmic site. This heteroplasmy affects the anticodon of a tRNA gene, allowing this single locus to function as a “dual” tRNA gene for two different amino acids. Here, we further explore the evolution of these unusual mitochondrial genomes by assembling complete mitochondrial sequences for two additional Oniscidean species, Trachelipus rathkei and Cylisticus convexus. Strikingly, we find evidence of two additional heteroplasmic sites that also alter tRNA anticodons, creating additional dual tRNA genes, and that are conserved across both species. These results suggest that the unique linear/circular organization of isopods’ mitochondrial genomes may facilitate the evolution of stable mitochondrial heteroplasmies, and, conversely, once such heteroplasmies have evolved, they constrain the multimeric structure of the mitochondrial genome in these species. Finally, we outline some possible future research directions to identify the factors influencing mitochondrial genome evolution in this group. PMID:25911226

  15. A story with a good ending: tRNA 3'-end maturation by CCA-adding enzymes.

    PubMed

    Xiong, Yong; Steitz, Thomas A

    2006-02-01

    CCA-adding enzymes (tRNA nucleotidyltransferases) are responsible for the maturation or repair of the functional 3' end of tRNAs. These enzymes are remarkable because they polymerize the essential nucleotides CCA onto the 3' terminus of tRNA precursors without using a nucleic acid template. Recent crystal structures, plus three decades of enzymology, have revealed the elegant mechanisms by which CCA-adding enzymes achieve their substrate specificity in a nucleic acid template independent fashion. The class I CCA-adding enzyme employs both an arginine sidechain and backbone phosphates of the bound tRNA to recognize incoming nucleotides. It switches from C to A addition through changes in the size and shape of the nucleotide-binding pocket, which is progressively altered by the elongating 3' terminus of the tRNA. By contrast, the class II CCA-adding enzyme uses only amino acid sidechains, which form a protein template for incoming nucleotide selection. PMID:16364630

  16. Identification of determinants for tRNA substrate recognition by Escherichia coli C/U34 2'-O-methyltransferase.

    PubMed

    Zhou, Mi; Long, Tao; Fang, Zhi-Peng; Zhou, Xiao-Long; Liu, Ru-Juan; Wang, En-Duo

    2015-01-01

    Post-transcriptional modifications bring chemical diversity to tRNAs, especially at positions 34 and 37 of the anticodon stem-loop (ASL). TrmL is the prokaryotic methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the wobble base of tRNA(Leu)CAA and tRNA(Leu)UAA isoacceptors. This Cm34/Um34 modification affects codon-anticodon interactions and is essential for translational fidelity. TrmL-catalyzed 2'-O-methylation requires its homodimerization; however, understanding of the tRNA recognition mechanism by TrmL remains elusive. In the current study, by measuring tRNA methylation by TrmL and performing kinetic analysis of tRNA mutants, we found that TrmL exhibits a fine-tuned tRNA substrate recognition mechanism. Anticodon stem-loop minihelices with an extension of 2 base pairs are the minimal substrate for EcTrmL methylation. A35 is a key residue for TrmL recognition, while A36-A37-A38 are important either via direct interaction with TrmL or due to the necessity for prior isopentenylation (i(6)) at A37. In addition, TrmL only methylates pyrimidines but not purine residues at the wobble position, and the 2'-O-methylation relies on prior N(6)-isopentenyladenosine modification at position 37. PMID:26106808

  17. Isolation of a class C transcription factor which forms a stable complex with tRNA genes.

    PubMed Central

    Ruet, A; Camier, S; Smagowicz, W; Sentenac, A; Fromageot, P

    1984-01-01

    A yeast extract was fractionated to resolve the factors involved in the transcription of yeast tRNA genes. An in vitro transcription system was reconstituted with two separate protein fractions and purified RNA polymerase C (III). Optimal conditions for tRNA synthesis have been determined. One essential component, termed tau factor, was partially purified by conventional chromatographic methods on heparin-agarose and DEAE-Sephadex; it sedimented as a large macromolecule in glycerol gradients (mol. wt. approximately 300 000). tau factor was found to form a stable complex with the tRNA gene in the absence of other transcriptional components. Complex formation is very fast, is not temperature dependent between 10 degrees C and 25 degrees C and does not require divalent cations. The factor-DNA complex is stable for at least 30 min at high salt concentration (0.1 M ammonium sulfate). These results indicate that gene recognition by a specific factor is a primary event in tRNA synthesis. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:6370678

  18. Archaeal proteins Nop10 and Gar1 increase the catalytic activity of Cbf5 in pseudouridylating tRNA.

    PubMed

    Kamalampeta, Rajashekhar; Kothe, Ute

    2012-01-01

    Cbf5 is a pseudouridine synthase that usually acts in a guide RNA-dependent manner as part of H/ACA small ribonucleoproteins; however archaeal Cbf5 can also act independently of guide RNA in modifying uridine 55 in tRNA. This guide-independent activity of Cbf5 is enhanced by proteins Nop10 and Gar1 which are also found in H/ACA small ribonucleoproteins. Here, we analyzed the specific contribution of Nop10 and Gar1 for Cbf5-catalyzed pseudouridylation of tRNA. Interestingly, both Nop10 and Gar1 not only increase Cbf5's affinity for tRNA, but they also directly enhance Cbf5's catalytic activity by increasing the k(cat) of the reaction. In contrast to the guide RNA-dependent reaction, Gar1 is not involved in product release after tRNA modification. These results in conjunction with structural information suggest that Nop10 and Gar1 stabilize Cbf5 in its active conformation; we hypothesize that this might also be true for guide-RNA dependent pseudouridine formation by Cbf5.

  19. Distinct modes of mature and precursor tRNA binding to Escherichia coli RNase P RNA revealed by NAIM analyses.

    PubMed Central

    Heide, C; Busch, S; Feltens, R; Hartmann, R K

    2001-01-01

    We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPalphaS (R = A, G, I) or Rp-c7-deaza-RMPalphaS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction. PMID:11345434

  20. Multiple Conserved Heteroplasmic Sites in tRNA Genes in the Mitochondrial Genomes of Terrestrial Isopods (Oniscidea).

    PubMed

    Chandler, Christopher H; Badawi, Myriam; Moumen, Bouziane; Grève, Pierre; Cordaux, Richard

    2015-04-24

    Mitochondrial genome structure and organization are relatively conserved among metazoans. However, in many isopods, especially the terrestrial isopods (Oniscidea), the mitochondrial genome consists of both ∼14-kb linear monomers and ∼28-kb circular dimers. This unusual organization is associated with an ancient and conserved constitutive heteroplasmic site. This heteroplasmy affects the anticodon of a tRNA gene, allowing this single locus to function as a "dual" tRNA gene for two different amino acids. Here, we further explore the evolution of these unusual mitochondrial genomes by assembling complete mitochondrial sequences for two additional Oniscidean species, Trachelipus rathkei and Cylisticus convexus. Strikingly, we find evidence of two additional heteroplasmic sites that also alter tRNA anticodons, creating additional dual tRNA genes, and that are conserved across both species. These results suggest that the unique linear/circular organization of isopods' mitochondrial genomes may facilitate the evolution of stable mitochondrial heteroplasmies, and, conversely, once such heteroplasmies have evolved, they constrain the multimeric structure of the mitochondrial genome in these species. Finally, we outline some possible future research directions to identify the factors influencing mitochondrial genome evolution in this group.

  1. Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding.

    PubMed

    Thrall, S H; Reinstein, J; Wöhrl, B M; Goody, R S

    1996-04-01

    A host cell-derived tRNA3Lys molecule is utilized by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to prime DNA synthesis from the viral RNA genome. We performed fluorescence titration experiments to characterize the interaction between RT and its natural primer, tRNA3Lys, and to address RT's putative role in the required and specific packaging of tRNA3Lys into the budding virus. Titration of RT with tRNA3Lys resulted in a 30% maximal quenching of RT tryptophan fluorescence, from which a dissociation constant (Kd) of 57.6 +/- 7.5 nM was derived. Titration of RT with Escherichia coli tRNA2Glu, E. coli tRNA2Tyr, E. coli tRNALys, yeast tRNAPhe, or in vitro-synthesized human tRNA3Lys (no base modifications) resulted in similar fluorescence changes and Kd values as obtained for the natural tRNA3Lys. The specific interaction between RT and tRNA3Lys during viral assembly suggested by previous in vivo studies is therefore not present in the fully processed, in vitro form of RT. Other factors during viral assembly must therefore cooperate in the packaging of tRNA3Lys. The nonspecific and ionic strength dependent RT-tRNA interaction detected in the present studies suggests that the overall shape and charges of tRNA constitute recognition features for RT binding. The fluorescence of the wyebutine base contained on the anticodon loop of yeast tRNAPhe was found to increase upon RT binding, supporting speculation that RT interacts with the anticodon loop of tRNA. The individual tRNAs also displaced a fluorescent DNA primer/template (p/t) substrate from RT, indicating overlapping tRNA and p/t binding sites. Cubic fit evaluation of the displacement titrations allowed further assessment of the affinities of the two competing ligands. The presence of both overlapping and separate p/t and tRNA binding regions on RT was tested by examination of the affinity of a possible RT bisubstrate type inhibitor, containing motifs proposed to be essential for both tRNA

  2. Timing and genesis of base-metal mineralisation in black shales of the Upper Permian Ravnefjeld Formation, Wegener Halvø, East Greenland

    NASA Astrophysics Data System (ADS)

    Pedersen, Mikael; Nielsen, Jesper K.; Boyce, Adrian J.; Fallick, Anthony E.

    2003-01-01

    Bituminous mud shales of the Upper Permian Ravnefjeld Formation (Zechstein 1 equivalent) are mineralised with zinc, lead and copper within a ca. 50 km2 area on Wegener Halvø in central East Greenland. The occurrence of base-metal sulphides in shale nodules cemented prior to compaction indicates an early commencement of base-metal mineralisation. In other cases, post-compactional sulphide textures are observed. Homogeneous lead isotope signatures of galena and sphalerite from the shales (206Pb/204Pb: 18.440-18.466; 207Pb/204Pb: 16.554-16.586; 208Pb/204Pb: 38.240-38.326) suggest that all base metals were introduced during a single hydrothermal event. Therefore, post-compactional textures are believed to result from recrystallisation of early diagenetic sulphides during deep burial in the Upper Cretaceous to Tertiary. Lead isotope signatures of galena hosted in Upper Permian carbonate build-ups are relatively heterogeneous compared to those of the shale-hosted sulphides. The observed relations indicate a shared lead source for the two types of mineralisation, but different degrees of homogenisation during mineralisation. This suggests that lead was introduced to the carbonate rocks and black shales during two separate events. δ34S of base-metal sulphides in the Ravnefjeld Formation lie between -12 and -4‰, whereas synsedimentary and early diagenetic pyrite in unmineralised shales in general have δ34S between -47 and -16.5‰. Early diagenetic pyrite in the Wegener Halvø area in general has δ34S 15 to 20‰ higher than the same pyrite morphotype in Triaselv in the western part of the basin. This relatively high δ34S can be explained by extensive microbial sulphate reduction within persistent euxinic (super-anoxic) bottom waters under which supply of isotopically light seawater sulphate (and disproportionation of intermediate sulphur compounds) was restricted. The sulphur in the base-metal sulphides is believed to represent sulphide-dominated pore water

  3. Duplication and Remolding of tRNA Genes in the Mitochondrial Genome of Reduvius tenebrosus (Hemiptera: Reduviidae).

    PubMed

    Jiang, Pei; Li, Hu; Song, Fan; Cai, Yao; Wang, Jianyun; Liu, Jinpeng; Cai, Wanzhi

    2016-01-01

    Most assassin bugs are predators that act as important natural enemies of insect pests. Mitochondrial (mt) genomes of these insects are double-strand circular DNAs that encode 37 genes. In the present study, we explore the duplication and rearrangement of tRNA genes in the mt genome of Reduvius tenebrosus, the first mt genome from the subfamily Reduviinae. The gene order rearranges from CR (control region)-trnI-trnQ-trnM-ND2 to CR-trnQ-trnI2-trnI1-trnM-ND2. We identified 23 tRNA genes, including 22 tRNAs commonly found in insects and an additional trnI (trnI2), which has high sequence similarity to trnM. We found several pseudo genes, such as pseudo-trnI, pseudo-CR, and pseudo-ND2, in the hotspot region of gene rearrangement (between the control region and ND2). These features provided evidence that this novel gene order could be explained by the tandem duplication/random loss (TDRL) model. The tRNA duplication/anticodon mutation mechanism further explains the presence of trnI2, which is remolded from a duplicated trnM in the TDRL process (through an anticodon mutation of CAT to GAT). Our study also raises new questions as to whether the two events proceed simultaneously and if the remolded tRNA gene is fully functional. Significantly, the duplicated tRNA gene in the mitochondrial genome has evolved independently at least two times within assassin bugs. PMID:27322247

  4. Duplication and Remolding of tRNA Genes in the Mitochondrial Genome of Reduvius tenebrosus (Hemiptera: Reduviidae)

    PubMed Central

    Jiang, Pei; Li, Hu; Song, Fan; Cai, Yao; Wang, Jianyun; Liu, Jinpeng; Cai, Wanzhi

    2016-01-01

    Most assassin bugs are predators that act as important natural enemies of insect pests. Mitochondrial (mt) genomes of these insects are double-strand circular DNAs that encode 37 genes. In the present study, we explore the duplication and rearrangement of tRNA genes in the mt genome of Reduvius tenebrosus, the first mt genome from the subfamily Reduviinae. The gene order rearranges from CR (control region)-trnI-trnQ-trnM-ND2 to CR-trnQ-trnI2-trnI1-trnM-ND2. We identified 23 tRNA genes, including 22 tRNAs commonly found in insects and an additional trnI (trnI2), which has high sequence similarity to trnM. We found several pseudo genes, such as pseudo-trnI, pseudo-CR, and pseudo-ND2, in the hotspot region of gene rearrangement (between the control region and ND2). These features provided evidence that this novel gene order could be explained by the tandem duplication/random loss (TDRL) model. The tRNA duplication/anticodon mutation mechanism further explains the presence of trnI2, which is remolded from a duplicated trnM in the TDRL process (through an anticodon mutation of CAT to GAT). Our study also raises new questions as to whether the two events proceed simultaneously and if the remolded tRNA gene is fully functional. Significantly, the duplicated tRNA gene in the mitochondrial genome has evolved independently at least two times within assassin bugs. PMID:27322247

  5. Insertion near the mitochondrial tyrosine tRNA gene in patients with mitochondrial diseases

    SciTech Connect

    Goto, Y.; Nonaka, I.; Horai, S.

    1994-09-01

    The 3243 mutation commonly found in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) has been occasionally detected in patients with chronic progressive external opthalmoplegia (CPEO). To elucidate the molecular mechanism underlying this phenomenon, an extensive mitochondrial (mt) DNA study was performed on such a patient (3243-CPEO). The newly discovered insertion was located in the noncoding region between cytrochrome c oxidase subunit 1 and tyrosine tRNA. The insertion was not found in 58 or 22 CPEO patients with or without mtDNA large-scale deletion but in another 3243-CPEO patient. In addition, the insertion was present in 1 of 116 normal Japanese, who had no 3243 mutation, and in 3 of 68 3243-MELAS patients. These results raise the possibility that the phenotypic expression of the 3243 mutation could be modulated or arranged by additional mtDNA mutations.

  6. tRNA Core Hypothesis for the Transition from the RNA World to the Ribonucleoprotein World

    PubMed Central

    de Farias, Savio T.; Rêgo, Thais G.; José, Marco V.

    2016-01-01

    Herein we present the tRNA core hypothesis, which emphasizes the central role of tRNAs molecules in the origin and evolution of fundamental biological processes. tRNAs gave origin to the first genes (mRNA) and the peptidyl transferase center (rRNA), proto-tRNAs were at the core of a proto-translation system, and the anticodon and operational codes then arose in tRNAs molecules. Metabolic pathways emerged from evolutionary pressures of the decoding systems. The transitions from the RNA world to the ribonucleoprotein world to modern biological systems were driven by three kinds of tRNAs transitions, to wit, tRNAs leading to both mRNA and rRNA. PMID:27023615

  7. Structure and in vitro transcription of a glycine tRNA gene from Bombyx mori.

    PubMed Central

    Fournier, A; Guérin, M A; Corlet, J; Clarkson, S G

    1984-01-01

    We report the sequences of a Bombyx mori tRNA1Gly gene, its flanking regions, and its in vitro transcription products. The 5' flanking DNA contains the sequences TATAC, TATTTT and TTC located 30, 18 and 4 nucleotides, respectively, in front of the transcription initiation site. These resemble, in both position and composition, sequences preceding other RNA polymerase III genes of B. mori. A deletion mutant retaining these conserved sequences and an additional 8 bp of flanking DNA is transcribed better than the wild-type gene in cell-free extracts from Xenopus laevis and B. mori. A mutant lacking the conserved sequences is expressed in the frog extract, but is inactive in the homologous system. Images Fig. 3. Fig. 5. PMID:6745242

  8. Detection of nucleoside Q precursor in methyl-deficient E.coli tRNA.

    PubMed Central

    Okada, N; Yasuda, T; Nishimura, S

    1977-01-01

    32P-Labeled tRNAAsn was isolated from methyl-deficient E. coli tRNA. Nucleotide sequence analysis showed that tRNAAsn contains three derivatives of the Q nucleoside, possibly Q precursors, in addition to guanosine in the first position of the anticodon. One of the Q precursors was isolated on a large scale. Its UV spectra were identical with those of normal Q, indicating that 7-deazaguanosine structure having a side chain at position C-7 is complete in the Q precursor. No radioactivity was incorporated into Q or Q precursors from either [methyl-14C]methionine, [1-14C]methionine or [U-14C]methionine, showing that methionine was not directly involved in the formation of Q. Images PMID:341083

  9. Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase

    PubMed Central

    Fang, Pengfei; Yu, Xue; Jeong, Seung Jae; Mirando, Adam; Chen, Kaige; Chen, Xin; Kim, Sunghoon; Francklyn, Christopher S.; Guo, Min

    2015-01-01

    The polyketide natural product borrelidin displays antibacterial, antifungal, antimalarial, anticancer, insecticidal and herbicidal activities through the selective inhibition of threonyl-tRNA synthetase (ThrRS). How borrelidin simultaneously attenuates bacterial growth and suppresses a variety of infections in plants and animals is not known. Here we show, using X-ray crystal structures and functional analyses, that a single molecule of borrelidin simultaneously occupies four distinct subsites within the catalytic domain of bacterial and human ThrRSs. These include the three substrate-binding sites for amino acid, ATP and tRNA associated with aminoacylation, and a fourth ‘orthogonal’ subsite created as a consequence of binding. Thus, borrelidin competes with all three aminoacylation substrates, providing a potent and redundant mechanism to inhibit ThrRS during protein synthesis. These results highlight a surprising natural design to achieve the quadrivalent inhibition of translation through a highly conserved family of enzymes. PMID:25824639

  10. On origin of genetic code and tRNA before translation

    PubMed Central

    2011-01-01

    Background Synthesis of proteins is based on the genetic code - a nearly universal assignment of codons to amino acids (aas). A major challenge to the understanding of the origins of this assignment is the archetypal "key-lock vs. frozen accident" dilemma. Here we re-examine this dilemma in light of 1) the fundamental veto on "foresight evolution", 2) modular structures of tRNAs and aminoacyl-tRNA synthetases, and 3) the updated library of aa-binding sites in RNA aptamers successfully selected in vitro for eight amino acids. Results The aa-binding sites of arginine, isoleucine and tyrosine contain both their cognate triplets, anticodons and codons. We have noticed that these cases might be associated with palindrome-dinucleotides. For example, one-base shift to the left brings arginine codons CGN, with CG at 1-2 positions, to the respective anticodons NCG, with CG at 2-3 positions. Formally, the concomitant presence of codons and anticodons is also expected in the reverse situation, with codons containing palindrome-dinucleotides at their 2-3 positions, and anticodons exhibiting them at 1-2 positions. A closer analysis reveals that, surprisingly, RNA binding sites for Arg, Ile and Tyr "prefer" (exactly as in the actual genetic code) the anticodon(2-3)/codon(1-2) tetramers to their anticodon(1-2)/codon(2-3) counterparts, despite the seemingly perfect symmetry of the latter. However, since in vitro selection of aa-specific RNA aptamers apparently had nothing to do with translation, this striking preference provides a new strong support to the notion of the genetic code emerging before translation, in response to catalytic (and possibly other) needs of ancient RNA life. Consistently with the pre-translation origin of the code, we propose here a new model of tRNA origin by the gradual, Fibonacci process-like, elongation of a tRNA molecule from a primordial coding triplet and 5'DCCA3' quadruplet (D is a base-determinator) to the eventual 76 base-long cloverleaf

  11. Insights into Folate/FAD-dependent tRNA Methyltransferase Mechanism

    PubMed Central

    Hamdane, Djemel; Argentini, Manuela; Cornu, David; Myllykallio, Hannu; Skouloubris, Stéphane; Hui-Bon-Hoa, Gaston; Golinelli-Pimpaneau, Béatrice

    2011-01-01

    The flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1- carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-l-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m5U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton. PMID:21846722

  12. Mitochondrial tRNA 5'-editing in Dictyostelium discoideum and Polysphondylium pallidum.

    PubMed

    Abad, Maria G; Long, Yicheng; Kinchen, R Dimitri; Schindel, Elinor T; Gray, Michael W; Jackman, Jane E

    2014-05-30

    Mitochondrial tRNA (mt-tRNA) 5'-editing was first described more than 20 years ago; however, the first candidates for 5'-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5'-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5'-editing in D. discoideum with 5'-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5'-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5'-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species. PMID:24737330

  13. Comparative analysis of dinoflagellate chloroplast genomes reveals rRNA and tRNA genes

    PubMed Central

    Barbrook, Adrian C; Santucci, Nicole; Plenderleith, Lindsey J; Hiller, Roger G; Howe, Christopher J

    2006-01-01

    Background Peridinin-containing dinoflagellates have a highly reduced chloroplast genome, which is unlike that found in other chloroplast containing organisms. Genome reduction appears to be the result of extensive transfer of genes to the nuclear genome. Unusually the genes believed to be remaining in the chloroplast genome are found on small DNA 'minicircles'. In this study we present a comparison of sets of minicircle sequences from three dinoflagellate species. Results PCR was used to amplify several minicircles from Amphidinium carterae so that a homologous set of gene-containing minicircles was available for Amphidinium carterae and Amphidinium operculatum, two apparently closely related peridinin-containing dinoflagellates. We compared the sequences of these minicircles to determine the content and characteristics of their chloroplast genomes. We also made comparisons with minicircles which had been obtained from Heterocapsa triquetra, another peridinin-containing dinoflagellate. These in silico comparisons have revealed several genetic features which were not apparent in single species analyses. The features include further protein coding genes, unusual rRNA genes, which we show are transcribed, and the first examples of tRNA genes from peridinin-containing dinoflagellate chloroplast genomes. Conclusion Comparative analysis of minicircle sequences has allowed us to identify previously unrecognised features of dinoflagellate chloroplast genomes, including additional protein and RNA genes. The chloroplast rRNA gene sequences are radically different from those in other organisms, and in many ways resemble the rRNA genes found in some highly reduced mitochondrial genomes. The retention of certain tRNA genes in the dinoflagellate chloroplast genome has important implications for models of chloroplast-mitochondrion interaction. PMID:17123435

  14. Mitochondrial tRNA 5′-Editing in Dictyostelium discoideum and Polysphondylium pallidum*

    PubMed Central

    Abad, Maria G.; Long, Yicheng; Kinchen, R. Dimitri; Schindel, Elinor T.; Gray, Michael W.; Jackman, Jane E.

    2014-01-01

    Mitochondrial tRNA (mt-tRNA) 5′-editing was first described more than 20 years ago; however, the first candidates for 5′-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5′-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5′-editing in D. discoideum with 5′-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5′-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5′-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species. PMID:24737330

  15. Formation of tRNA granules in the nucleus of heat-induced human cells

    SciTech Connect

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

  16. tRNA is a new target for cleavage by a MazF toxin.

    PubMed

    Schifano, Jason M; Cruz, Jonathan W; Vvedenskaya, Irina O; Edifor, Regina; Ouyang, Ming; Husson, Robert N; Nickels, Bryce E; Woychik, Nancy A

    2016-02-18

    Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNA(Pro14) D-loop or within the tRNA(Lys43) anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth. PMID:26740583

  17. tRNA is a new target for cleavage by a MazF toxin

    PubMed Central

    Schifano, Jason M.; Cruz, Jonathan W.; Vvedenskaya, Irina O.; Edifor, Regina; Ouyang, Ming; Husson, Robert N.; Nickels, Bryce E.; Woychik, Nancy A.

    2016-01-01

    Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNAPro14 D-loop or within the tRNALys43 anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth. PMID:26740583

  18. Simulating movement of tRNA through the ribosome during hybrid-state formation

    NASA Astrophysics Data System (ADS)

    Whitford, Paul C.; Sanbonmatsu, Karissa Y.

    2013-09-01

    Biomolecular simulations provide a means for exploring the relationship between flexibility, energetics, structure, and function. With the availability of atomic models from X-ray crystallography and cryoelectron microscopy (cryo-EM), and rapid increases in computing capacity, it is now possible to apply molecular dynamics (MD) simulations to large biomolecular machines, and systematically partition the factors that contribute to function. A large biomolecular complex for which atomic models are available is the ribosome. In the cell, the ribosome reads messenger RNA (mRNA) in order to synthesize proteins. During this essential process, the ribosome undergoes a wide range of conformational rearrangements. One of the most poorly understood transitions is translocation: the process by which transfer RNA (tRNA) molecules move between binding sites inside of the ribosome. The first step of translocation is the adoption of a "hybrid" configuration by the tRNAs, which is accompanied by large-scale rotations in the ribosomal subunits. To illuminate the relationship between these rearrangements, we apply MD simulations using a multi-basin structure-based (SMOG) model, together with targeted molecular dynamics protocols. From 120 simulated transitions, we demonstrate the viability of a particular route during P/E hybrid-state formation, where there is asynchronous movement along rotation and tRNA coordinates. These simulations not only suggest an ordering of events, but they highlight atomic interactions that may influence the kinetics of hybrid-state formation. From these simulations, we also identify steric features (H74 and surrounding residues) encountered during the hybrid transition, and observe that flexibility of the single-stranded 3'-CCA tail is essential for it to reach the endpoint. Together, these simulations provide a set of structural and energetic signatures that suggest strategies for modulating the physical-chemical properties of protein synthesis by the

  19. A Drosophila model for mito-nuclear diseases generated by an incompatible interaction between tRNA and tRNA synthetase

    PubMed Central

    Holmbeck, Marissa A.; Donner, Julia R.; Villa-Cuesta, Eugenia; Rand, David M.

    2015-01-01

    ABSTRACT Communication between the mitochondrial and nuclear genomes is vital for cellular function. The assembly of mitochondrial enzyme complexes, which produce the majority of cellular energy, requires the coordinated expression and translation of both mitochondrially and nuclear-encoded proteins. The joint genetic architecture of this system complicates the basis of mitochondrial diseases, and mutations both in mitochondrial DNA (mtDNA)- and nuclear-encoded genes have been implicated in mitochondrial dysfunction. Previously, in a set of mitochondrial-nuclear introgression strains, we characterized a dual genome epistasis in which a naturally occurring mutation in the Drosophila simulans simw501 mtDNA-encoded transfer RNA (tRNA) for tyrosine (tRNATyr) interacts with a mutation in the nuclear-encoded mitochondrially localized tyrosyl-tRNA synthetase from Drosophila melanogaster. Here, we show that the incompatible mitochondrial-nuclear combination results in locomotor defects, reduced mitochondrial respiratory capacity, decreased oxidative phosphorylation (OXPHOS) enzyme activity and severe alterations in mitochondrial morphology. Transgenic rescue strains containing nuclear variants of the tyrosyl-tRNA synthetase are sufficient to rescue many of the deleterious phenotypes identified when paired with the simw501 mtDNA. However, the severity of this defective mito-nuclear interaction varies across traits and genetic backgrounds, suggesting that the impact of mitochondrial dysfunction might be tissue specific. Because mutations in mitochondrial tRNATyr are associated with exercise intolerance in humans, this mitochondrial-nuclear introgression model in Drosophila provides a means to dissect the molecular basis of these, and other, mitochondrial diseases that are a consequence of the joint genetic architecture of mitochondrial function. PMID:26035388

  20. A Drosophila model for mito-nuclear diseases generated by an incompatible interaction between tRNA and tRNA synthetase.

    PubMed

    Holmbeck, Marissa A; Donner, Julia R; Villa-Cuesta, Eugenia; Rand, David M

    2015-08-01

    Communication between the mitochondrial and nuclear genomes is vital for cellular function. The assembly of mitochondrial enzyme complexes, which produce the majority of cellular energy, requires the coordinated expression and translation of both mitochondrially and nuclear-encoded proteins. The joint genetic architecture of this system complicates the basis of mitochondrial diseases, and mutations both in mitochondrial DNA (mtDNA)- and nuclear-encoded genes have been implicated in mitochondrial dysfunction. Previously, in a set of mitochondrial-nuclear introgression strains, we characterized a dual genome epistasis in which a naturally occurring mutation in the Drosophila simulans simw(501) mtDNA-encoded transfer RNA (tRNA) for tyrosine (tRNA(Tyr)) interacts with a mutation in the nuclear-encoded mitochondrially localized tyrosyl-tRNA synthetase from Drosophila melanogaster. Here, we show that the incompatible mitochondrial-nuclear combination results in locomotor defects, reduced mitochondrial respiratory capacity, decreased oxidative phosphorylation (OXPHOS) enzyme activity and severe alterations in mitochondrial morphology. Transgenic rescue strains containing nuclear variants of the tyrosyl-tRNA synthetase are sufficient to rescue many of the deleterious phenotypes identified when paired with the simw(501) mtDNA. However, the severity of this defective mito-nuclear interaction varies across traits and genetic backgrounds, suggesting that the impact of mitochondrial dysfunction might be tissue specific. Because mutations in mitochondrial tRNA(Tyr) are associated with exercise intolerance in humans, this mitochondrial-nuclear introgression model in Drosophila provides a means to dissect the molecular basis of these, and other, mitochondrial diseases that are a consequence of the joint genetic architecture of mitochondrial function. PMID:26035388

  1. Biophysical insights into the intercalative interaction of surfactant cobalt(III) complexes of certain diimine ligands bound to yeast tRNA: Effects of hydrophobicity.

    PubMed

    Nagaraj, Karuppiah; Sakthinathan, Subramanian; Arunachalam, Sankaralingam

    2015-08-01

    The interaction of two surfactant cobalt(III) complexes, cis-[Co(ip)₂(DA)₂](ClO₄)₃ 1 and cis-[Co(dpq)₂(DA)₂](ClO₄)₃ 2 where ip=imidazo[4,5-f][1,10]phenanthroline and dpq=dipyrido[3,2-d:2'-3'-f]quinoxaline with yeast tRNA have been explored by using electronic absorption, competitive binding, electrochemical studies and viscosity measurements. The results suggest that these complexes can bind to tRNA by intercalation. The presence of hydrophobic diimine ligand and the long aliphatic double chains of these complexes facilitate its intercalative interaction with the hydrophobic interior of the tRNA. The extent of tRNA binding of complex 2 has greater affinity than that of complex containing imidazo[4,5-f][1,10]phenanthroline ligands.

  2. Biophysical insights into the intercalative interaction of surfactant cobalt(III) complexes of certain diimine ligands bound to yeast tRNA: Effects of hydrophobicity

    NASA Astrophysics Data System (ADS)

    Nagaraj, Karuppiah; Sakthinathan, Subramanian; Arunachalam, Sankaralingam

    2015-08-01

    The interaction of two surfactant cobalt(III) complexes, cis-[Co(ip)2(DA)2](ClO4)3 1 and cis-[Co(dpq)2(DA)2](ClO4)3 2 where ip = imidazo[4,5-f][1,10]phenanthroline and dpq = dipyrido[3,2-d:2‧-3‧-f]quinoxaline with yeast tRNA have been explored by using electronic absorption, competitive binding, electrochemical studies and viscosity measurements. The results suggest that these complexes can bind to tRNA by intercalation. The presence of hydrophobic diimine ligand and the long aliphatic double chains of these complexes facilitate its intercalative interaction with the hydrophobic interior of the tRNA. The extent of tRNA binding of complex 2 has greater affinity than that of complex containing imidazo[4,5-f][1,10]phenanthroline ligands.

  3. Avatar pre-tRNAs help elucidate the properties of tRNA-splicing endonucleases that produce tRNA from permuted genes.

    PubMed

    Tocchini-Valentini, Giuseppe D; Tocchini-Valentini, Glauco P

    2012-12-26

    Unusual tRNA genes, found in some algae, have their mature terminal 3' portion in front of their 5' portion in the genome. The transcripts from such genes must be cleaved by a pre-tRNA endonuclease to form a functional tRNA. We present a mechanism for the generation of "corrected" tRNAs from such a "permuted" pre-tRNA configuration. We used two avatar (av) or model pre-tRNAs and two splicing endonucleases with distinct mechanisms of recognition of the pre-tRNA. The splicing results are compatible with an evolutionary route in which permuted genes result from a duplication event followed by DNA rearrangement. The model pre-tRNAs permit description of the features that a transcript, derived from a rearranged duplicated gene, must have to give rise to functional tRNA. The two tRNA endonucleases are a eukaryal enzyme that normally acts in a mature domain-dependent mode and an archaeal enzyme that acts in a mature domain-independent mode. Both av pre-tRNAs are able to fold into two conformations: 1 and 2. We find that only conformation 2 can yield a corrected functional tRNA. This result is consistent with contemporary algae representing snapshots of different evolutionary stages, with duplicated genes preceding recombinatorial events generating a permutated gene. In a scenario elucidated by the use of the av pre-tRNAs, algal permuted tRNA genes could have further lost one of two mature domains, eliminating steric problems for the algal tRNA endonuclease, which remains a typical eukaryal enzyme capable of correcting the permuted transcript to a functional tRNA.

  4. Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5' Leader Sequences.

    PubMed

    Niland, Courtney N; Zhao, Jing; Lin, Hsuan-Chun; Anderson, David R; Jankowsky, Eckhard; Harris, Michael E

    2016-08-19

    Maturation of tRNA depends on a single endonuclease, ribonuclease P (RNase P), to remove highly variable 5' leader sequences from precursor tRNA transcripts. Here, we use high-throughput enzymology to report multiple-turnover and single-turnover kinetics for Escherichia coli RNase P processing of all possible 5' leader sequences, including nucleotides contacting both the RNA and protein subunits of RNase P. The results reveal that the identity of N(-2) and N(-3) relative to the cleavage site at N(1) primarily control alternative substrate selection and act at the level of association not the cleavage step. As a consequence, the specificity for N(-1), which contacts the active site and contributes to catalysis, is suppressed. This study demonstrates high-throughput RNA enzymology as a means to globally determine RNA specificity landscapes and reveals the mechanism of substrate discrimination by a widespread and essential RNA-processing enzyme. PMID:27336323

  5. Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5' Leader Sequences.

    PubMed

    Niland, Courtney N; Zhao, Jing; Lin, Hsuan-Chun; Anderson, David R; Jankowsky, Eckhard; Harris, Michael E

    2016-08-19

    Maturation of tRNA depends on a single endonuclease, ribonuclease P (RNase P), to remove highly variable 5' leader sequences from precursor tRNA transcripts. Here, we use high-throughput enzymology to report multiple-turnover and single-turnover kinetics for Escherichia coli RNase P processing of all possible 5' leader sequences, including nucleotides contacting both the RNA and protein subunits of RNase P. The results reveal that the identity of N(-2) and N(-3) relative to the cleavage site at N(1) primarily control alternative substrate selection and act at the level of association not the cleavage step. As a consequence, the specificity for N(-1), which contacts the active site and contributes to catalysis, is suppressed. This study demonstrates high-throughput RNA enzymology as a means to globally determine RNA specificity landscapes and reveals the mechanism of substrate discrimination by a widespread and essential RNA-processing enzyme.

  6. The use of 5'-phospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA.

    PubMed Central

    Robertson, S A; Noren, C J; Anthony-Cahill, S J; Griffith, M C; Schultz, P G

    1989-01-01

    Methodology is described for the synthesis and chemical aminoacylation of the hybrid dinucleotide 5'-phospho-2'-deoxyribocytidylylriboadenosine (pdCpA). Ligation of aminoacylated pdCpA to a truncated amber suppressor tRNACUA (-CA) using T4 RNA ligase generates an aminoacylated suppressor tRNA which can be used for site-specific incorporation of unnatural amino acids into proteins. Both the ligation and in vitro suppression efficiencies are the same when either pCpA or pdCpA is used. The use of deoxycytidine simplifies the chemistry involved in the synthesis of the dinucleotide pCpA. In addition, these results demonstrate that ribocytidine is not required for recognition of the aminoacylated tRNA during protein synthesis. PMID:2602139

  7. Crystal Structure of Staphylococcus aureus tRNA Adenosine Deaminase TadA in Complex with RNA

    SciTech Connect

    Losey,H.; Ruthenburg, A.; Verdine, G.

    2006-01-01

    Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.

  8. Peptides from aminoacyl-tRNA synthetases can cure the defects due to mutations in mt tRNA genes.

    PubMed

    Francisci, Silvia; Montanari, Arianna; De Luca, Cristina; Frontali, Laura

    2011-11-01

    Recent results from several laboratories have confirmed that human and yeast leucyl- and valyl-tRNA synthetases can rescue the respiratory defects due to mutations in mitochondrial tRNA genes. In this report we show that this effect cannot be ascribed to the catalytic activity per se and that isolated domains of aminoacyl-tRNA synthetases and even short peptides thereof have suppressing effects.

  9. A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase—extensive moonlighting in mitochondrial tRNA biogenesis

    PubMed Central

    Vilardo, Elisa; Nachbagauer, Christa; Buzet, Aurélie; Taschner, Andreas; Holzmann, Johann; Rossmanith, Walter

    2012-01-01

    Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5′ extensions, is the methyltransferase responsible for m1G9 and m1A9 formation. The ability of the mitochondrial tRNA:m1R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5′ end processing. PMID:23042678

  10. The initiator methionine tRNA drives cell migration and invasion leading to increased metastatic potential in melanoma

    PubMed Central

    Birch, Joanna; Clarke, Cassie J.; Campbell, Andrew D.; Campbell, Kirsteen; Mitchell, Louise; Liko, Dritan; Kalna, Gabriela; Strathdee, Douglas; Sansom, Owen J.; Neilson, Matthew; Blyth, Karen

    2016-01-01

    ABSTRACT The cell's repertoire of transfer RNAs (tRNAs) has been linked to cancer. Recently, the level of the initiator methionine tRNA (tRNAiMet) in stromal fibroblasts has been shown to influence extracellular matrix (ECM) secretion to drive tumour growth and angiogenesis. Here we show that increased tRNAiMet within cancer cells does not influence tumour growth, but drives cell migration and invasion via a mechanism that is independent from ECM synthesis and dependent on α5β1 integrin and levels of the translation initiation ternary complex. In vivo and ex vivo migration (but not proliferation) of melanoblasts is significantly enhanced in transgenic mice which express additional copies of the tRNAiMet gene. We show that increased tRNAiMet in melanoma drives migratory, invasive behaviour and metastatic potential without affecting cell proliferation and primary tumour growth, and that expression of RNA polymerase III-associated genes (which drive tRNA expression) are elevated in metastases by comparison with primary tumours. Thus, specific alterations to the cancer cell tRNA repertoire drive a migration/invasion programme that may lead to metastasis. PMID:27543055

  11. Phosphorylation of Elp1 by Hrr25 Is Required for Elongator-Dependent tRNA Modification in Yeast

    PubMed Central

    Abdel-Fattah, Wael; Jablonowski, Daniel; Di Santo, Rachael; Thüring, Kathrin L.; Scheidt, Viktor; Hammermeister, Alexander; ten Have, Sara; Helm, Mark; Schaffrath, Raffael; Stark, Michael J. R.

    2015-01-01

    Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase. PMID:25569479

  12. Single sequence of a helix-loop peptide confers functional anticodon recognition on two tRNA synthetases.

    PubMed Central

    Auld, D S; Schmimmel, P

    1996-01-01

    The specific aminoacylation of RNA oligonucleotides whose sequences are based on the acceptor stems of tRNAs can be viewed as an operational RNA code for amino acids that may be related to the development of the genetic code. Many synthetases also have direct interactions with tRNA anticodon triplets and, in some cases, these interactions are thought to be essential for aminoacylation specificity. In these instances, an unresolved question is whether interactions with parts of the tRNA outside of the anticodon are sufficient for decoding genetic information. Escherichia coli isoleucyl- and methionyl-tRNA synthetases are closely related enzymes that interact with their respective anticodons. We used binary combinatorial mutagenesis of a 10 amino acid anticodon binding peptide in these two enzymes to identify composite sequences that would confer function to both enzymes despite their recognizing different anticodons. A single peptide was found that confers function to both enzymes in vivo and in vitro. Thus, even in enzymes where anticodon interactions are normally important for distinguishing one tRNA from another, these interactions can be 'neutralized' without losing specificity of amino-acylation. We suggest that acceptor helix interactions may play a role in providing the needed specificity. Images PMID:8605884

  13. Trying on tRNA for Size: RNase P and the T-box Riboswitch as Molecular Rulers

    PubMed Central

    Zhang, Jinwei; Ferré-DAmaré, Adrian R.

    2016-01-01

    Length determination is a fundamental problem in biology and chemistry. Numerous proteins measure distances on linear biopolymers to exert effects with remarkable spatial precision. Recently, ruler-like devices made of noncoding RNAs have been structurally and biochemically characterized. Two prominent examples are the RNase P ribozyme and the T-box riboswitch. Both act as molecular calipers. The two RNAs clamp onto the elbow of tRNA (or pre-tRNA) and make distance measurements orthogonal to each other. Here, we compare and contrast the molecular ruler characteristics of these RNAs. RNase P appears pre-configured to measure a fixed distance on pre-tRNA to ensure the fidelity of its maturation. RNase P is a multiple-turnover ribozyme, and its rigid structure efficiently selects pre-tRNAs, cleaves, and releases them. In contrast, the T-box is flexible and segmented, an architecture that adapts to the intrinsically flexible tRNA. The tripartite T-box inspects the overall shape, anticodon sequence, and aminoacylation status of an incoming tRNA while it folds co-transcriptionally, leading to a singular, conditional genetic switching event. The elucidation of the structures and mechanisms of action of these two RNA molecular rulers may augur the discovery of new RNA measuring devices in noncoding and viral transcriptomes, and inform the design of artificial RNA rulers. PMID:27043647

  14. Specific phase arrest of cell cycle restores cell viability against tRNA cleavage by killer toxin.

    PubMed

    Shigematsu, Megumi; Ogawa, Tetsuhiro; Kitamoto, Hiroko K; Hidaka, Makoto; Masaki, Haruhiko

    2012-04-20

    Zymocin and PaT are killer toxins that induce cell cycle arrest of sensitive yeast cells in G1 and S phase, respectively. Recent studies have revealed that these two toxins cleave specific tRNAs, indicating that the cell growth impairment is due to the tRNA cleavage. Additionally, we have previously shown that the active domain of colicin D (D-CRD), which also cleaves specific Escherichia coli tRNAs, statically impairs growth when expressed in yeast cells. To verify that phase-specific cell cycle arrest is also induced by the expression of D-CRD, D-CRD and the subunits of zymocin and PaT that have tRNA cleaving activity were expressed in yeast cells and cell cycle status was analyzed. Our results indicate that phase-specific arrest does not commonly occur by tRNA cleavage, and it saves the cell viability. Furthermore, the extent of protein synthesis impairment may determine the phase specificity of cell cycle arrest. PMID:22450321

  15. The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis

    PubMed Central

    Tuorto, Francesca; Herbst, Friederike; Alerasool, Nader; Bender, Sebastian; Popp, Oliver; Federico, Giuseppina; Reitter, Sonja; Liebers, Reinhard; Stoecklin, Georg; Gröne, Hermann-Josef; Dittmar, Gunnar; Glimm, Hanno; Lyko, Frank

    2015-01-01

    The Dnmt2 enzyme utilizes the catalytic mechanism of eukaryotic DNA methyltransferases to methylate several tRNAs at cytosine 38. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile, and the biological function of Dnmt2 has remained elusive. Here, we show that endochondral ossification is delayed in newborn Dnmt2-deficient mice, which is accompanied by a reduction of the haematopoietic stem and progenitor cell population and a cell-autonomous defect in their differentiation. RNA bisulfite sequencing revealed that Dnmt2 methylates C38 of tRNA AspGTC, GlyGCC, and ValAAC, thus preventing tRNA fragmentation. Proteomic analyses from primary bone marrow cells uncovered systematic differences in protein expression that are due to specific codon mistranslation by tRNAs lacking Dnmt2-dependent methylation. Our observations demonstrate that Dnmt2 plays an important role in haematopoiesis and define a novel function of C38 tRNA methylation in the discrimination of near-cognate codons, thereby ensuring accurate polypeptide synthesis. PMID:26271101

  16. Trying on tRNA for Size: RNase P and the T-box Riboswitch as Molecular Rulers.

    PubMed

    Zhang, Jinwei; Ferré-DAmaré, Adrian R

    2016-01-01

    Length determination is a fundamental problem in biology and chemistry. Numerous proteins measure distances on linear biopolymers to exert effects with remarkable spatial precision. Recently, ruler-like devices made of noncoding RNAs have been structurally and biochemically characterized. Two prominent examples are the RNase P ribozyme and the T-box riboswitch. Both act as molecular calipers. The two RNAs clamp onto the elbow of tRNA (or pre-tRNA) and make distance measurements orthogonal to each other. Here, we compare and contrast the molecular ruler characteristics of these RNAs. RNase P appears pre-configured to measure a fixed distance on pre-tRNA to ensure the fidelity of its maturation. RNase P is a multiple-turnover ribozyme, and its rigid structure efficiently selects pre-tRNAs, cleaves, and releases them. In contrast, the T-box is flexible and segmented, an architecture that adapts to the intrinsically flexible tRNA. The tripartite T-box inspects the overall shape, anticodon sequence, and aminoacylation status of an incoming tRNA while it folds co-transcriptionally, leading to a singular, conditional genetic switching event. The elucidation of the structures and mechanisms of action of these two RNA molecular rulers may augur the discovery of new RNA measuring devices in noncoding and viral transcriptomes, and inform the design of artificial RNA rulers. PMID:27043647

  17. MD Simulations of tRNA and Aminoacyl-tRNA Synthetases: Dynamics, Folding, Binding, and Allostery

    PubMed Central

    Li, Rongzhong; Macnamara, Lindsay M.; Leuchter, Jessica D.; Alexander, Rebecca W.; Cho, Samuel S.

    2015-01-01

    While tRNA and aminoacyl-tRNA synthetases are classes of biomolecules that have been extensively studied for decades, the finer details of how they carry out their fundamental biological functions in protein synthesis remain a challenge. Recent molecular dynamics (MD) simulations are verifying experimental observations and providing new insight that cannot be addressed from experiments alone. Throughout the review, we briefly discuss important historical events to provide a context for how far the field has progressed over the past few decades. We then review the background of tRNA molecules, aminoacyl-tRNA synthetases, and current state of the art MD simulation techniques for those who may be unfamiliar with any of those fields. Recent MD simulations of tRNA dynamics and folding and of aminoacyl-tRNA synthetase dynamics and mechanistic characterizations are discussed. We highlight the recent successes and discuss how important questions can be addressed using current MD simulations techniques. We also outline several natural next steps for computational studies of AARS:tRNA complexes. PMID:26184179

  18. Crystal structure of a 4-thiouridine synthetase–RNA complex reveals specificity of tRNA U8 modification

    PubMed Central

    Neumann, Piotr; Naumann, Peter-Thomas; Erwin, Whitney M.; Lauhon, Charles T.; Ficner, Ralf

    2014-01-01

    In prokaryotes and archaea transfer ribonucleic acid (tRNA) stability as well as cellular UV protection relies on the post-transcriptional modification of uracil at position 8 (U8) of tRNAs by the 4-thiouridine synthetase ThiI. Here, we report three crystal structures of ThiI from Thermotoga maritima in complex with a truncated tRNA. The RNA is mainly bound by the N-terminal ferredoxin-like domain (NFLD) and the THUMP domain of one subunit within the ThiI homo-dimer thereby positioning the U8 close to the catalytic center in the pyrophosphatase domain of the other subunit. The recognition of the 3’-CCA end by the THUMP domain yields a molecular ruler defining the specificity for U8 thiolation. This first structure of a THUMP/NFLD-RNA complex might serve as paradigm for the RNA recognition by THUMP domains of other proteins. The ternary ThiI–RNA–ATP complex shows no significant structural changes due to adenosine triphosphate (ATP) binding, but two different states of active site loops are observed independent of the nucleotide loading state. Thereby conformational changes of the active site are coupled with conformational changes of the bound RNA. The ThiI–RNA complex structures indicate that full-length tRNA has to adopt a non-canonical conformation upon binding to ThiI. PMID:24705700

  19. Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure

    PubMed Central

    Létoquart, Juliette; van Tran, Nhan; Caroline, Vonny; Aleksandrov, Alexey; Lazar, Noureddine; van Tilbeurgh, Herman; Liger, Dominique; Graille, Marc

    2015-01-01

    Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm5U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity. PMID:26438534

  20. The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway.

    PubMed

    Hasler, Daniele; Lehmann, Gerhard; Murakawa, Yasuhiro; Klironomos, Filippos; Jakob, Leonhard; Grässer, Friedrich A; Rajewsky, Nikolaus; Landthaler, Markus; Meister, Gunter

    2016-07-01

    The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading. PMID:27345152

  1. Connecting the kinetics and energy landscape of tRNA translocation on the ribosome.

    PubMed

    Whitford, Paul C; Blanchard, Scott C; Cate, Jamie H D; Sanbonmatsu, Karissa Y

    2013-01-01

    Functional rearrangements in biomolecular assemblies result from diffusion across an underlying energy landscape. While bulk kinetic measurements rely on discrete state-like approximations to the energy landscape, single-molecule methods can project the free energy onto specific coordinates. With measures of the diffusion, one may establish a quantitative bridge between state-like kinetic measurements and the continuous energy landscape. We used an all-atom molecular dynamics simulation of the 70S ribosome (2.1 million atoms; 1.3 microseconds) to provide this bridge for specific conformational events associated with the process of tRNA translocation. Starting from a pre-translocation configuration, we identified sets of residues that collectively undergo rotary rearrangements implicated in ribosome function. Estimates of the diffusion coefficients along these collective coordinates for translocation were then used to interconvert between experimental rates and measures of the energy landscape. This analysis, in conjunction with previously reported experimental rates of translocation, provides an upper-bound estimate of the free-energy barriers associated with translocation. While this analysis was performed for a particular kinetic scheme of translocation, the quantitative framework is general and may be applied to energetic and kinetic descriptions that include any number of intermediates and transition states.

  2. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B

    PubMed Central

    Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  3. Interaction of elongation factor 1 with aminoacylated brome mosaic virus and tRNA's.

    PubMed Central

    Bastin, M; Hall, T C

    1976-01-01

    Tyrosylated Brome mosaic virus RNA was found to interact with a binary complex of wheat germ, elongation factor 1 and [3H]GTP. Increasing amounts of the aminoacylated viral RNA proportionately reduced radioactivity bound to a nitrocellulose filter, as has previously been noted by others for the charged forms of tobacco mosaic virus, turnip yellow mosaic virus, and tRNA's. However, Sephadex chromatography of the products showed that instead of forming the ternary complex elongation factor-GTP-aminoacyl RNA, the viral RNA caused release of GTP from its complex with elongation factor. Acetylated tyrosyl Brome mosaic virus RNA did not react with the binary complex,and only a slight degree, if any, of stabilization of tyrosine bound to viral RNA was observed after interaction with elongation factor 1. Although such interactions are similar to the reaction of elongation factor with aminoacyl-tRNA , the release of GTP is different and accentuates the possible role for aminoacylation in transcription rather than in translation events. PMID:978788

  4. The selective tRNA aminoacylation mechanism based on a single G•U pair.

    PubMed

    Naganuma, Masahiro; Sekine, Shun-ichi; Chong, Yeeting Esther; Guo, Min; Yang, Xiang-Lei; Gamper, Howard; Hou, Ya-Ming; Schimmel, Paul; Yokoyama, Shigeyuki

    2014-06-26

    Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNA(Ala) selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNA(Ala) with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and the major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3'-CCA region. Thus, the 3'-CCA region of tRNA(Ala) with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.

  5. A Mutant Trna Affects δ-Mediated Transcription in Saccharomyces Cerevisiae

    PubMed Central

    Happel, A. M.; Winston, F.

    1992-01-01

    Mutations in the SPT3, SPT7, SPT8 and SPT15 genes define one class of trans-acting mutations that are strong suppressors of insertion mutations caused by Ty elements or by the Ty long terminal repeat sequence, δ. These SPT genes are required for normal transcription of Ty elements, and their gene products are believed to be involved in initiation of Ty transcription from δ sequences. We have isolated and analyzed extragenic suppressors of spt3 mutations. These new mutations, named rsp, partially suppress the requirement for SPT3, SPT7, SPT8 and SPT15 functions. In addition, rsp mutations cause changes in transcription of some δ insertions in an SPT(+) genetic background. Interactions between mutations in the four identified RSP genes show a number of interesting genetic properties, including the failure of unlinked rsp mutations to complement for recessive phenotypes. Cloning and sequencing of one rsp mutant gene, rsp4-27, showed that it encodes a frameshift suppressor glycine tRNA. Our results indicate that the other three RSP genes also encode frameshift suppressor glycine tRNAs. In addition, other types of frameshift suppressor glycine tRNAs can confer some Rsp(-) phenotypes. PMID:1330824

  6. Formation of tRNA granules in the nucleus of heat-induced human cells.

    PubMed

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-02-01

    The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA(Met) (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA(Met) was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules. PMID:22244871

  7. RNA helicase module in an acetyltransferase that modifies a specific tRNA anticodon

    PubMed Central

    Chimnaronk, Sarin; Suzuki, Tateki; Manita, Tetsuhiro; Ikeuchi, Yoshiho; Yao, Min; Suzuki, Tsutomu; Tanaka, Isao

    2009-01-01

    Post-transcriptional RNA modifications in the anticodon of transfer RNAs frequently contribute to the high fidelity of protein synthesis. In eubacteria, two genome-encoded transfer RNA (tRNA) species bear the same CAU sequence as the anticodons, which are differentiated by modified cytidines at the wobble positions. The elongator tRNAMet accepts an acetyl moiety at the wobble base to form N4-acetylcytidine (ac4C): an inherent modification ensures precise decoding of the AUG codon by strengthening C−G base-pair interaction and concurrently preventing misreading of the near cognate AUA codon. We have determined the crystal structure of tRNAMet cytidine acetyltransferase (TmcA) from Escherichia coli complexed with two natural ligands, acetyl-CoA and ADP, at 2.35 Å resolution. The structure unexpectedly reveals an idiosyncratic RNA helicase module fused with a GCN5-related N-acetyltransferase (GNAT) fold, which intimately cross-interact. Taken together with the biochemical evidence, we further unravelled the function of acetyl-CoA as an enzyme-activating switch, and propose that an RNA helicase motor driven by ATP hydrolysis is used to deliver the wobble base to the active centre of the GNAT domain. PMID:19322199

  8. Limited diagnostic value of enzyme analysis in patients with mitochondrial tRNA mutations.

    PubMed

    Wibrand, Flemming; Jeppesen, Tina D; Frederiksen, Anja L; Olsen, David B; Duno, Morten; Schwartz, Marianne; Vissing, John

    2010-05-01

    We evaluated the diagnostic value of respiratory chain (RC) enzyme analysis of muscle in adult patients with mitochondrial myopathy (MM). RC enzyme activity was measured in muscle biopsies from 39 patients who carry either the 3243A>G mutation, other tRNA point mutations, or single, large-scale deletions of mtDNA. Findings were compared with those obtained from asymptomatic relatives with the 3243A>G mutation, myotonic dystrophy patients, and healthy subjects. Plasma lactate concentration, maximal oxygen uptake, and ragged-red fibers/cytochrome c-negative fibers in muscle were also determined. Only 10% of patients with the 3243A>G point mutation had decreased enzyme activity of one or more RC complexes, whereas this was the case for 83% of patients with other point mutations and 62% of patients with deletions. Abnormal muscle histochemistry was found in 65%, 100%, and 85% of patients, respectively, in these three groups. The results indicate that RC enzyme analysis in muscle is not a sensitive test for MM in adults. In these patients, abnormal muscle histochemistry appears to be a better predictor ofMM.

  9. Crystal structures of the bifunctional tRNA methyltransferase Trm5a

    PubMed Central

    Wang, Caiyan; Jia, Qian; Chen, Ran; Wei, Yuming; Li, Juntao; Ma, Jie; Xie, Wei

    2016-01-01

    tRNA methyltransferase Trm5 catalyses the transfer of a methyl group from S-adenosyl-L-methionine to G37 in eukaryotes and archaea. The N1-methylated guanosine is the product of the initial step of the wyosine hypermodification, which is essential for the maintenance of the reading frame during translation. As a unique member of this enzyme family, Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4-demethylwyosine at position 37 to produce isowyosine, but the mechanism for the double methylation is poorly understood. Here we report four crystal structures of PaTrm5a ranging from 1.7- to 2.3-Å, in the apo form or in complex with various SAM analogues. These structures reveal that Asp243 specifically recognises the base moiety of SAM at the active site. Interestingly, the protein in our structures all displays an extended conformation, quite different from the well-folded conformation of Trm5b from Methanocaldococcus jannaschii reported previously, despite their similar overall architectures. To rule out the possibilities of crystallisation artefacts, we conducted the fluorescence resonance energy transfer (FRET) experiments. The FRET data suggested that PaTrm5a adopts a naturally extended conformation in solution, and therefore the open conformation is a genuine state of PaTrm5a. PMID:27629654

  10. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B.

    PubMed

    Wemhoff, Sabrina; Klassen, Roland; Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  11. Nonrecombinant Meiosis I Nondisjunction in Saccharomyces Cerevisiae Induced by Trna Ochre Suppressors

    PubMed Central

    Louis, E. J.; Haber, J. E.

    1989-01-01

    The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink. PMID:2680759

  12. Crystal structures of the bifunctional tRNA methyltransferase Trm5a.

    PubMed

    Wang, Caiyan; Jia, Qian; Chen, Ran; Wei, Yuming; Li, Juntao; Ma, Jie; Xie, Wei

    2016-01-01

    tRNA methyltransferase Trm5 catalyses the transfer of a methyl group from S-adenosyl-L-methionine to G37 in eukaryotes and archaea. The N1-methylated guanosine is the product of the initial step of the wyosine hypermodification, which is essential for the maintenance of the reading frame during translation. As a unique member of this enzyme family, Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4-demethylwyosine at position 37 to produce isowyosine, but the mechanism for the double methylation is poorly understood. Here we report four crystal structures of PaTrm5a ranging from 1.7- to 2.3-Å, in the apo form or in complex with various SAM analogues. These structures reveal that Asp243 specifically recognises the base moiety of SAM at the active site. Interestingly, the protein in our structures all displays an extended conformation, quite different from the well-folded conformation of Trm5b from Methanocaldococcus jannaschii reported previously, despite their similar overall architectures. To rule out the possibilities of crystallisation artefacts, we conducted the fluorescence resonance energy transfer (FRET) experiments. The FRET data suggested that PaTrm5a adopts a naturally extended conformation in solution, and therefore the open conformation is a genuine state of PaTrm5a. PMID:27629654

  13. Origin and evolution of group I introns in cyanobacterial tRNA genes.

    PubMed Central

    Paquin, B; Kathe, S D; Nierzwicki-Bauer, S A; Shub, D A

    1997-01-01

    Many tRNA(Leu)UAA genes from plastids contain a group I intron. An intron is also inserted in the same gene at the same position in cyanobacteria, the bacterial progenitors of plastids, suggesting an ancient bacterial origin for this intron. A group I intron has also been found in the tRNA(fMet) gene of some cyanobacteria but not in plastids, suggesting a more recent origin for this intron. In this study, we investigate the phylogenetic distributions of the two introns among cyanobacteria, from the earliest branching to the more derived species. The phylogenetic distribution of the tRNA(Leu)UAA intron follows the clustering of rRNA sequences, being either absent or present in clades of closely related species, with only one exception in the Pseudanabaena group. Our data support the notion that the tRNA(Leu)UAA intron was inherited by cyanobacteria and plastids through a common ancestor. Conversely, the tRNA(fMet) intron has a sporadic distribution, implying that many gains and losses occurred during cyanobacterial evolution. Interestingly, a phylogenetic tree inferred from intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history. PMID:9352932

  14. tRNA modified bases and oxidative stress in Salmonella typhimurium

    SciTech Connect

    Kramer, G.F.

    1987-01-01

    The mechanisms of toxicity of two different environmental stresses have been characterized in Salmonella typhimurium. The toxicity of near-UV (NUV) light (300-400 nm) appeared to be mediated by oxidative mechanisms. The overproduction of NUV-absorbing proteins sensitized the cells to killing by NUV. Selenium also appeared to be toxic to S. typhimurium by oxidative mechanisms. At low concentrations, the main target for this toxicity appeared to be intracellular thiols. At higher concentrations, selenite toxicity appeared to have been mediated by oxygen radicals which we have shown to be produced by the reactions of selenite with sulfhydryl groups. Such radicals may also have been involved in the selenite mutagenicity we have observed in S. typhimurium. The function of two different modified bases with respect to such oxidative stress has been characterized. The isolation of mutants lacking these bases has facilitated this investigation. S. typhimurium contained a single seleno-modified base, 5-methylaminomethyl-2-selenouridine (mnm{sup 5}Se{sup 2}U). Mutants which were unable to incorporate selenium into their tRNA (selA) were isolated based on a pleiotropic defect in selenium metabolism.

  15. The role of mitochondrial tRNA mutations in lung cancer

    PubMed Central

    Wang, Lie; Chen, Zhi-Jun; Zhang, Yong-Kui; Le, Han-Bo

    2015-01-01

    Alternations in mitochondrial genome resulting in mitochondrial dysfunction have long been hypothesized to be involved in tumorigenesis. Mitochondrial tRNA (mt-tRNA) is known for its high frequencies of polymorphisms and mutations, however, the roles of these mutations and polymorphisms in lung cancer are among heated debates. To evaluate the possible roles of reported mt-tRNA mutations in lung cancer, we examine recent published paper concerning three mt-tRNA mutations with lung cancer: A7460G in tRNASer (UCN) gene, G5563A in tRNATrp gene and A12172G in tRNAHis gene. We perform the phylogenetic approach to investigate the deleterious roles of these mutations in lung cancer, moreover, we use bioinformatics tool to predict the secondary structure of mt-tRNAs with and without these mutations. In addition, through the application of pathogenicity scoring system, we find that only the A12172G mutation is regarded as a pathogenic mutation, whereas other mutations may act as neutral polymorphisms in human population. Thus, our study provides the novel insight into the molecular pathogenesis of mt-tRNA mutations in lung cancer. PMID:26550263

  16. Hypoxic stress suppresses RNA polymerase III recruitment and tRNA gene transcription in cardiomyocytes

    PubMed Central

    Ernens, Isabelle; Goodfellow, Sarah J.; Innes, Fiona; Kenneth, Niall S.; Derblay, Louise E.; White, Robert J.; Scott, Pamela H.

    2006-01-01

    RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes. PMID:16407335

  17. Single-Turnover Kinetics of Methyl Transfer to tRNA by Methyltransferases

    PubMed Central

    Hou, Ya-Ming

    2016-01-01

    Summary Methyl transfer from S-adenosyl methionine (abbreviated as AdoMet) to biologically active molecules such as mRNAs and tRNAs is one of the most fundamental and widespread reactions in nature, occurring in all three domains of life. The measurement of kinetic constants of AdoMet-dependent methyl transfer is therefore important for understanding the reaction mechanism in the context of biology. When kinetic constants of methyl transfer are measured in steady state over multiple rounds of turnover, the meaning of these constants is difficult to define and is often limited by non-chemical steps of the reaction, such as product release after each turnover. Here the measurement of kinetic constants of methyl transfer by tRNA methyltransferases in rapid equilibrium binding condition for one methyl transfer is described. The advantage of such a measurement is that the meaning of kinetic constants can be directly assigned to the steps associated with the chemistry of methyl transfer, including the substrate binding affinity to the methyl transferase, the pre-chemistry re-arrangement of the active site, and the chemical step of methyl transfer. An additional advantage is that kinetic constants measured for one methyl transfer can be correlated with structural information of the methyl transferase to gain direct insight into its reaction mechanism. PMID:26965259

  18. Biosynthesis of Threonylcarbamoyl Adenosine (t6A), a Universal tRNA Nucleoside*

    PubMed Central

    Deutsch, Christopher; El Yacoubi, Basma; de Crécy-Lagard, Valérie; Iwata-Reuyl, Dirk

    2012-01-01

    The anticodon stem-loop (ASL) of transfer RNAs (tRNAs) drives decoding by interacting directly with the mRNA through codon/anticodon pairing. Chemically complex nucleoside modifications found in the ASL at positions 34 or 37 are known to be required for accurate decoding. Although over 100 distinct modifications have been structurally characterized in tRNAs, only a few are universally conserved, among them threonylcarbamoyl adenosine (t6A), found at position 37 in the anticodon loop of a subset of tRNA. Structural studies predict an important role for t6A in translational fidelity, and in vivo work supports this prediction. Although pioneering work in the 1970s identified the fundamental substrates for t6A biosynthesis, the enzymes responsible for its biosynthesis have remained an enigma. We report here the discovery that in bacteria four proteins (YgjD, YrdC, YjeE, and YeaZ) are both necessary and sufficient for t6A biosynthesis in vitro. Notably, YrdC and YgjD are members of universally conserved families that were ranked among the top 10 proteins of unknown function in need of functional characterization, while YeaZ and YjeE are specific to bacteria. This latter observation, coupled with the essentiality of all four proteins in bacteria, establishes this pathway as a compelling new target for antimicrobial development. PMID:22378793

  19. The Enzymatic Paradox of Yeast Arginyl-tRNA Synthetase: Exclusive Arginine Transfer Controlled by a Flexible Mechanism of tRNA Recognition

    PubMed Central

    Eriani, Gilbert; Geslain, Renaud

    2016-01-01

    Identity determinants are essential for the accurate recognition of transfer RNAs by aminoacyl-tRNA synthetases. To date, arginine determinants in the yeast Saccharomyces cerevisiae have been identified exclusively in vitro and only on a limited number of tRNA Arginine isoacceptors. In the current study, we favor a full cellular approach and expand the investigation of arginine determinants to all four tRNA Arg isoacceptors. More precisely, this work scrutinizes the relevance of the tRNA nucleotides at position 20, 35 and 36 in the yeast arginylation reaction. We built 21 mutants by site-directed mutagenesis and tested their functionality in YAL5, a previously engineered yeast knockout deficient for the expression of tRNA Arg CCG. Arginylation levels were also monitored using Northern blot. Our data collected in vivo correlate with previous observations. C35 is the prominent arginine determinant followed by G36 or U36 (G/U36). In addition, although there is no major arginine determinant in the D loop, the recognition of tRNA Arg ICG relies to some extent on the nucleotide at position 20. This work refines the existing model for tRNA Arg recognition. Our observations indicate that yeast Arginyl-tRNA synthetase (yArgRS) relies on distinct mechanisms to aminoacylate the four isoacceptors. Finally, according to our refined model, yArgRS is able to accommodate tRNA Arg scaffolds presenting N34, C/G35 and G/A/U36 anticodons while maintaining specificity. We discuss the mechanistic and potential physiological implications of these findings. PMID:26844776

  20. Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons.

    PubMed

    Sakai, Yusuke; Miyauchi, Kenjyo; Kimura, Satoshi; Suzuki, Tsutomu

    2016-01-29

    Post-transcriptional modifications at the anticodon first (wobble) position of tRNA play critical roles in precise decoding of genetic codes. 5-carboxymethoxyuridine (cmo(5)U) and its methyl ester derivative 5-methoxycarbonylmethoxyuridine (mcmo(5)U) are modified nucleosides found at the anticodon wobble position in several tRNAs from Gram-negative bacteria. cmo(5)U and mcmo(5)U facilitate non-Watson-Crick base pairing with guanosine and pyrimidines at the third positions of codons, thereby expanding decoding capabilities. By mass spectrometric analyses of individual tRNAs and a shotgun approach of total RNA from Escherichia coli, we identified mcmo(5)U as a major modification in tRNA(Ala1), tRNA(Ser1), tRNA(Pro3) and tRNA(Thr4); by contrast, cmo(5)U was present primarily in tRNA(Leu3) and tRNA(Val1). In addition, we discovered 5-methoxycarbonylmethoxy-2'-O-methyluridine (mcmo(5)Um) as a novel but minor modification in tRNA(Ser1). Terminal methylation frequency of mcmo(5)U in tRNA(Pro3) was low (≈30%) in the early log phase of cell growth, gradually increased as growth proceeded and reached nearly 100% in late log and stationary phases. We identified CmoM (previously known as SmtA), an AdoMet-dependent methyltransferase that methylates cmo(5)U to form mcmo(5)U. A luciferase reporter assay based on a +1 frameshift construct revealed that terminal methylation of mcmo(5)U contributes to the decoding ability of tRNA(Ala1). PMID:26681692

  1. The Roles of Compensatory Evolution and Constraint in Aminoacyl tRNA Synthetase Evolution.

    PubMed

    Adrion, Jeffrey R; White, P Signe; Montooth, Kristi L

    2016-01-01

    Mitochondrial protein translation requires interactions between transfer RNAs encoded by the mitochondrial genome (mt-tRNAs) and mitochondrial aminoacyl tRNA synthetase proteins (mt-aaRS) encoded by the nuclear genome. It has been argued that animal mt-tRNAs have higher deleterious substitution rates relative to their nuclear-encoded counterparts, the cytoplasmic tRNAs (cyt-tRNAs). This dynamic predicts elevated rates of compensatory evolution of mt-aaRS that interact with mt-tRNAs, relative to aaRS that interact with cyt-tRNAs (cyt-aaRS). We find that mt-aaRS do evolve at significantly higher rates (exemplified by higher dN and dN/dS) relative to cyt-aaRS, across mammals, birds, and Drosophila. While this pattern supports a model of compensatory evolution, the level at which a gene is expressed is a more general predictor of protein evolutionary rate. We find that gene expression level explains 10-56% of the variance in aaRS dN/dS, and that cyt-aaRS are more highly expressed in addition to having lower dN/dS values relative to mt-aaRS, consistent with more highly expressed genes being more evolutionarily constrained. Furthermore, we find no evidence of positive selection acting on either class of aaRS protein, as would be expected under a model of compensatory evolution. Nevertheless, the signature of faster mt-aaRS evolution persists in mammalian, but not bird or Drosophila, lineages after controlling for gene expression, suggesting some additional effect of compensatory evolution for mammalian mt-aaRS. We conclude that gene expression is the strongest factor governing differential amino acid substitution rates in proteins interacting with mitochondrial versus cytoplasmic factors, with important differences in mt-aaRS molecular evolution among taxonomic groups.

  2. Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons

    PubMed Central

    Sakai, Yusuke; Miyauchi, Kenjyo; Kimura, Satoshi; Suzuki, Tsutomu

    2016-01-01

    Post-transcriptional modifications at the anticodon first (wobble) position of tRNA play critical roles in precise decoding of genetic codes. 5-carboxymethoxyuridine (cmo5U) and its methyl ester derivative 5-methoxycarbonylmethoxyuridine (mcmo5U) are modified nucleosides found at the anticodon wobble position in several tRNAs from Gram-negative bacteria. cmo5U and mcmo5U facilitate non-Watson–Crick base pairing with guanosine and pyrimidines at the third positions of codons, thereby expanding decoding capabilities. By mass spectrometric analyses of individual tRNAs and a shotgun approach of total RNA from Escherichia coli, we identified mcmo5U as a major modification in tRNAAla1, tRNASer1, tRNAPro3 and tRNAThr4; by contrast, cmo5U was present primarily in tRNALeu3 and tRNAVal1. In addition, we discovered 5-methoxycarbonylmethoxy-2′-O-methyluridine (mcmo5Um) as a novel but minor modification in tRNASer1. Terminal methylation frequency of mcmo5U in tRNAPro3 was low (≈30%) in the early log phase of cell growth, gradually increased as growth proceeded and reached nearly 100% in late log and stationary phases. We identified CmoM (previously known as SmtA), an AdoMet-dependent methyltransferase that methylates cmo5U to form mcmo5U. A luciferase reporter assay based on a +1 frameshift construct revealed that terminal methylation of mcmo5U contributes to the decoding ability of tRNAAla1. PMID:26681692

  3. Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification.

    PubMed

    Payne, Karl A P; Fisher, Karl; Sjuts, Hanno; Dunstan, Mark S; Bellina, Bruno; Johannissen, Linus; Barran, Perdita; Hay, Sam; Rigby, Stephen E J; Leys, David

    2015-11-13

    Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5'-GUN-3' tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes. PMID:26378237

  4. Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification*

    PubMed Central

    Payne, Karl A. P.; Fisher, Karl; Sjuts, Hanno; Dunstan, Mark S.; Bellina, Bruno; Johannissen, Linus; Barran, Perdita; Hay, Sam; Rigby, Stephen E. J.; Leys, David

    2015-01-01

    Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5′-GUN-3′ tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes. PMID:26378237

  5. A Hypertension-Associated tRNAAla Mutation Alters tRNA Metabolism and Mitochondrial Function

    PubMed Central

    Jiang, Pingping; Wang, Meng; Xue, Ling; Xiao, Yun; Yu, Jialing; Wang, Hui; Yao, Juan; Liu, Hao; Peng, Yanyan; Liu, Hanqing; Li, Haiying; Chen, Ye

    2016-01-01

    In this report, we investigated the pathophysiology of a novel hypertension-associated mitochondrial tRNAAla 5655A → G (m.5655A → G) mutation. The destabilization of a highly conserved base pairing (A1-U72) at the aminoacyl acceptor stem by an m.5655A → G mutation altered the tRNAAla function. An in vitro processing analysis showed that the m.5655A → G mutation reduced the efficiency of tRNAAla precursor 5′ end cleavage catalyzed by RNase P. By using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mitochondrial DNA (mtDNA)-less (ρo) cells, we showed a 41% reduction in the steady-state level of tRNAAla in mutant cybrids. The mutation caused an improperly aminoacylated tRNAAla, as suggested by aberrantly aminoacylated tRNAAla and slower electrophoretic mobility of mutated tRNA. A failure in tRNAAla metabolism contributed to variable reductions in six mtDNA-encoded polypeptides in mutant cells, ranging from 21% to 37.5%, with an average of a 29.1% reduction, compared to levels of the controls. The impaired translation caused reduced activities of mitochondrial respiration chains. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These caused increases in the production of reactive oxygen species in the mutant cybrids. The data provide evidence for the association of the tRNAAla 5655A → G mutation with hypertension. PMID:27161322

  6. Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNA

    SciTech Connect

    Hountondji, C.; Schmitter, J.M.; Beauvallet, C.; Blanquet, S.

    1987-08-25

    Periodate-oxidized tRNA/sup Phe/ (tRNA/sub ox//sup Phe/) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the ..cap alpha../sub 2/..beta../sub 2/ enzyme with tRNA/sub ox//sup Phe/ results in the loss of tRNA/sup Phe/ aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-(/sup 14/C)tRNA/sub ox//sup Phe/ covalent complex indicates that the large (..cap alpha.., M/sub r/ 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA/sub ox//sup Phe/. The (/sup 14/C)tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys(Ado)-Phe, Ala-Asp-Lys(Ado)-Leu, and Lys-Ile-Lys(Ado)-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the ..cap alpha.. subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases.

  7. Developmental roles of Drosophila tRNA processing endonuclease RNase ZL as revealed with a conditional rescue system

    PubMed Central

    Xie, Xie; Dubrovskaya, Veronica; Yacoub, Nancy; Walska, Joanna; Gleason, Tara; Reid, Katherine; Dubrovsky, Edward B.

    2013-01-01

    Drosophila RNase ZL (dRNaseZ) belongs to a family of endoribonucleases with a major role in tRNA 3′-end processing. The biochemical function of RNase ZL is conserved from yeast to human. Here we present a study of its biological function during Drosophila development. In flies, dRNaseZ provides a non-redundant function, as the RNZED24 knockout (KO) mutation causes early larval lethality. Mosaic and conditional rescue techniques were employed to determine dRNaseZ requirements at later stages. We found that dRNaseZ activity is essential for all phases of fly development that involve cell division, including growth of adult tissue progenitors during larval and metamorphic stages, and gametogenesis in adults. At the cellular level, two major phenotypes were identified – cell growth deficiency in endoreplicating tissues and cell cycle arrest in mitotic tissues. While cell growth and proliferation are both dependant on protein synthesis, the two phenotypes displayed reliance on different dRNaseZ functions. We found that dRNaseZ KO completely blocks tRNA maturation without diminishing the abundance of mature tRNA molecules. Our data indicate that growth arrest of endoreplicating cells is primarily attributed to the relocation of the pool of mature tRNAs into the nuclei causing a decrease in translation efficiency. Mitotically dividing cells appear to be less dependent on translation machinery as they maintain their normal size when deprived of dRNaseZ activity, but rather display a cell cycle arrest at the G2-M transition. PMID:23867108

  8. Purine N7 groups that are crucial to the interaction of Escherichia coli rnase P RNA with tRNA.

    PubMed Central

    Heide, C; Feltens, R; Hartmann, R K

    2001-01-01

    We have detected by nucleotide analog interference mapping (NAIM) purine N7 functional groups in Escherichia coli RNase P RNA that are important for tRNA binding under moderate salt conditions (0.1 M Mg2+, 0.1 M NH4+). The majority of identified positions represent highly or universally conserved nucleotides. Our assay system allowed us, for the first time, to identify c7-deaza interference effects at two G residues (G292, G306). Several c7-deazaadenine interference effects (A62, A65, A136, A249, A334, A351) have also been identified in other studies performed at very different salt concentrations, either selecting for substrate binding in the presence of 0.025 M Ca2+ and 1 M NH4+ or self-cleavage of a ptRNA-RNase P RNA conjugate in the presence of 3 M NH4+ or Na+. This indicates that these N7 functional groups play a key role in the structural organization of ribozyme-substrate and -product complexes. We further observed that a c7-deaza modification at A76 of tRNA interferes with tRNA binding to and ptRNA processing by E. coli RNase P RNA. This finding combined with the strong c7-deaza interference at G292 of RNase P RNA supports a model in which substrate and product binding to E. coli RNase P RNA involves the formation of intermolecular base triples (A258-G292-C75 and G291-G259-A76). PMID:11453068

  9. Preparation of an ochre suppressor tRNA recognizing exclusively UAA codon by using the molecular surgery technique.

    PubMed

    Yokogawa, Takashi; Hassan, Hanim Munirah Binti Che; Yokota, Yukiko; Ohno, Satoshi; Nishikawa, Kazuya

    2009-01-01

    In order to create an ochre suppressor tRNA which exclusively recognizes UAA codon, we replaced the G34 at the first position of yeast tRNA(Tyr)[GPsiA] anticodon with pseudouridine34 (Psi34) by using the molecular surgery technique. This tRNA(Tyr)[PsiPsiA] recognized only the UAA codon as expectedly, but tRNA(Tyr)[UPsiA] made as a control also behaved similarly. This result may suggest that U34 must be somehow modified to facilitate the wobble-pairing to G at the third position of codon. PMID:19749377

  10. New features of Asian Crassostrea oyster mitochondrial genomes: a novel alloacceptor tRNA gene recruitment and two novel ORFs.

    PubMed

    Wu, Xiangyun; Li, Xiaoling; Li, Lu; Xu, Xiaodong; Xia, Jianjun; Yu, Ziniu

    2012-10-10

    A feasible way to perform evolutionary analyses is to compare characters divergent enough to observe significant differences, but sufficiently similar to exclude saturation of the differences that occurred. Thus, comparisons of invertebrate mitochondrial (mt) genomes at low taxonomic levels can be extremely helpful in investigating patterns of variation and evolutionary dynamics of genomes, as intermediate stages of the process may be identified. Fortunately, in this study, we newly sequenced the mt genome of the eighth member of Asian Crassostrea oysters which can provide necessary intermediate characters for us to believe that the variation of Crassostrea mt genomes is considerably greater than previously acknowledged. Several new features of Asian Crassostrea oyster mitochondrial genomes were revealed, and our results are particularly significant as they 1) suggest a novel model of alloacceptor tRNA gene recruitment, namely "vertical" tRNA gene recruitment, which can be successfully used to explain the origination of the unusually additional trnK and trnQ genes (annotated as trnK(2) and trnQ(2) respectively) in the mt genomes of the five Asian oysters, and we speculate that this recruitment progress may be a common phenomenon in the evolution of the tRNA multigene family; 2) reveal the existence of two additional, lineage-specific, mtDNA-encoded genes that may originate from duplication of nad2 followed by rapid evolutionary change. Each of these two genes encodes a unique amino terminal signal peptide, thus each might possess an unknown function; and 3) identify for the first time the atp8 gene in oysters. The present study thus gives further credence to the comparison of congeneric bivalves as a meaningful strategy to investigate mt genomic evolutionary trends in genome organization, tRNA multigene family, and gene loss and/or duplication that are difficult to undertake at higher taxonomic levels. In particular, our study provides new evidence for the

  11. C/D box sRNA, CRISPR RNA and tRNA processing in an archaeon with a minimal fragmented genome.

    PubMed

    Richter, Hagen; Mohr, Sabine; Randau, Lennart

    2013-02-01

    The analysis of deep sequencing data allows for a genome-wide overview of all the small RNA molecules (the 'sRNome') that are present in a single organism. In the present paper, we review the processing of CRISPR (clustered regularly interspaced short palindromic repeats) RNA, C/D box sRNA (small non-coding RNA) and tRNA in Nanoarchaeum equitans. The minimal and fragmented genome of this tiny archaeon permits a sequencing depth that enables the identification of processing intermediates in the study of RNA processing pathways. These intermediates include circular C/D box sRNA molecules and tRNA half precursors.

  12. Analysis of the 3′ ends of tRNA as the cause of insertion sites of foreign DNA in Prochlorococcus *

    PubMed Central

    Liu, Hai-lan; Zhu, Jun

    2010-01-01

    The purpose of this study was to investigate the characteristics of transfer RNA (tRNA) responsible for the association between tRNA genes and genes of apparently foreign origin (genomic islands) in five high-light adapted Prochlorococcus strains. Both bidirectional best BLASTP (basic local alignment search tool for proteins) search and the conservation of gene order against each other were utilized to identify genomic islands, and 7 genomic islands were found to be immediately adjacent to tRNAs in Prochlorococcus marinus AS9601, 11 in P. marinus MIT9515, 8 in P. marinus MED4, 6 in P. marinus MIT9301, and 6 in P. marinus MIT9312. Monte Carlo simulation showed that tRNA genes are hotspots for the integration of genomic islands in Prochlorococcus strains. The tRNA genes associated with genomic islands showed the following characteristics: (1) the association was biased towards a specific subset of all iso-accepting tRNA genes; (2) the codon usages of genes within genomic islands appear to be unrelated to the codons recognized by associated tRNAs; and, (3) the majority of the 3′ ends of associated tRNAs lack CCA ends. These findings contradict previous hypotheses concerning the molecular basis for the frequent use of tRNA as the insertion site for foreign genetic materials. The analysis of a genomic island associated with a tRNA-Asn gene in P. marinus MIT9301 suggests that foreign genetic material is inserted into the host genomes by means of site-specific recombination, with the 3′ end of the tRNA as the target, and during the process, a direct repeat of the 3′ end sequence of a boundary tRNA (namely, a scar from the process of insertion) is formed elsewhere in the genomic island. Through the analysis of the sequences of these targets, it can be concluded that a region characterized by both high GC content and a palindromic structure is the preferred insertion site. PMID:20803775

  13. Specific replacement of Q base in the anticodon of tRNA by guanine catalyzed by a cell-free extract of rabbit reticulocytes.

    PubMed Central

    Okada, N; Harada, F; Nishimura, S

    1976-01-01

    Guanylation of tRNA by a lysate of rabbit reticulocytes was reported previously by Farkas and Singh. This reaction was investigated further using 18 purified E. coli tRNAs as acceptors.Results showed that only tRNATyr, tRNAHis, tRNAAsn and tRNAAsp which contain the modified nucleoside Q in the anticodon acted as acceptors. Analysis of the nucleotide sequences in the guanylated tRNA showed that guanine specifically replaced Q base in these tRNAs. Images PMID:792816

  14. Three-Dimensional Algebraic Models of the tRNA Code and 12 Graphs for Representing the Amino Acids.

    PubMed

    José, Marco V; Morgado, Eberto R; Guimarães, Romeu Cardoso; Zamudio, Gabriel S; de Farías, Sávio Torres; Bobadilla, Juan R; Sosa, Daniela

    2014-01-01

    Three-dimensional algebraic models, also called Genetic Hotels, are developed to represent the Standard Genetic Code, the Standard tRNA Code (S-tRNA-C), and the Human tRNA code (H-tRNA-C). New algebraic concepts are introduced to be able to describe these models, to wit, the generalization of the 2n-Klein Group and the concept of a subgroup coset with a tail. We found that the H-tRNA-C displayed broken symmetries in regard to the S-tRNA-C, which is highly symmetric. We also show that there are only 12 ways to represent each of the corresponding phenotypic graphs of amino acids. The averages of statistical centrality measures of the 12 graphs for each of the three codes are carried out and they are statistically compared. The phenotypic graphs of the S-tRNA-C display a common triangular prism of amino acids in 10 out of the 12 graphs, whilst the corresponding graphs for the H-tRNA-C display only two triangular prisms. The graphs exhibit disjoint clusters of amino acids when their polar requirement values are used. We contend that the S-tRNA-C is in a frozen-like state, whereas the H-tRNA-C may be in an evolving state. PMID:25370377

  15. Structural and functional insights into tRNA binding and adenosine N1-methylation by an archaeal Trm10 homologue

    PubMed Central

    Van Laer, Bart; Roovers, Martine; Wauters, Lina; Kasprzak, Joanna M.; Dyzma, Michal; Deyaert, Egon; Kumar Singh, Ranjan; Feller, André; Bujnicki, Janusz M.; Droogmans, Louis; Versées, Wim

    2016-01-01

    Purine nucleosides on position 9 of eukaryal and archaeal tRNAs are frequently modified in vivo by the post-transcriptional addition of a methyl group on their N1 atom. The methyltransferase Trm10 is responsible for this modification in both these domains of life. While certain Trm10 orthologues specifically methylate either guanosine or adenosine at position 9 of tRNA, others have a dual specificity. Until now structural information about this enzyme family was only available for the catalytic SPOUT domain of Trm10 proteins that show specificity toward guanosine. Here, we present the first crystal structure of a full length Trm10 orthologue specific for adenosine, revealing next to the catalytic SPOUT domain also N- and C-terminal domains. This structure hence provides crucial insights in the tRNA binding mechanism of this unique monomeric family of SPOUT methyltransferases. Moreover, structural comparison of this adenosine-specific Trm10 orthologue with guanosine-specific Trm10 orthologues suggests that the N1 methylation of adenosine relies on additional catalytic residues. PMID:26673726

  16. Three-Dimensional Algebraic Models of the tRNA Code and 12 Graphs for Representing the Amino Acids

    PubMed Central

    José, Marco V.; Morgado, Eberto R.; Guimarães, Romeu Cardoso; Zamudio, Gabriel S.; de Farías, Sávio Torres; Bobadilla, Juan R.; Sosa, Daniela

    2014-01-01

    Three-dimensional algebraic models, also called Genetic Hotels, are developed to represent the Standard Genetic Code, the Standard tRNA Code (S-tRNA-C), and the Human tRNA code (H-tRNA-C). New algebraic concepts are introduced to be able to describe these models, to wit, the generalization of the 2n-Klein Group and the concept of a subgroup coset with a tail. We found that the H-tRNA-C displayed broken symmetries in regard to the S-tRNA-C, which is highly symmetric. We also show that there are only 12 ways to represent each of the corresponding phenotypic graphs of amino acids. The averages of statistical centrality measures of the 12 graphs for each of the three codes are carried out and they are statistically compared. The phenotypic graphs of the S-tRNA-C display a common triangular prism of amino acids in 10 out of the 12 graphs, whilst the corresponding graphs for the H-tRNA-C display only two triangular prisms. The graphs exhibit disjoint clusters of amino acids when their polar requirement values are used. We contend that the S-tRNA-C is in a frozen-like state, whereas the H-tRNA-C may be in an evolving state. PMID:25370377

  17. Conserved sequences in both coding and 5' flanking regions of mammalian opal suppressor tRNA genes.

    PubMed Central

    Pratt, K; Eden, F C; You, K H; O'Neill, V A; Hatfield, D

    1985-01-01

    The rabbit genome encodes an opal suppressor tRNA gene. The coding region is strictly conserved between the rabbit gene and the corresponding gene in the human genome. The rabbit opal suppressor gene contains the consensus sequence in the 3' internal control region but like the human and chicken genes, the rabbit 5' internal control region contains two additional nucleotides. The 5' flanking sequences of the rabbit and the human opal suppressor genes contain extensive regions of homology. A subset of these homologies is also present 5' to the chicken opal suppressor gene. Both the rabbit and the human genomes also encode a pseudogene. That of the rabbit lacks the 3' half of the coding region. Neither pseudogene has homologous regions to the 5' flanking regions of the genes. The presence of 5' homologies flanking only the transcribed genes and not the pseudogenes suggests that these regions may be regulatory control elements specifically involved in the expression of the eukaryotic opal suppressor gene. Moreover the strict conservation of coding sequences indicates functional importance for the opal suppressor tRNA genes. Images PMID:4022772

  18. Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

    PubMed Central

    Au, Hilda H.; Cornilescu, Gabriel; Mouzakis, Kathryn D.; Ren, Qian; Burke, Jordan E.; Lee, Seonghoon; Butcher, Samuel E.; Jan, Eric

    2015-01-01

    The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame. PMID:26554019

  19. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis.

  20. A role for [Fe4S4] clusters in tRNA recognition—a theoretical study

    PubMed Central

    Stiebritz, Martin T.

    2014-01-01

    Over the past several years, structural studies have led to the unexpected discovery of iron–sulfur clusters in enzymes that are involved in DNA replication/repair and protein biosynthesis. Although these clusters are generally well-studied cofactors, their significance in the new contexts often remains elusive. One fascinating example is a tryptophanyl-tRNA synthetase from the thermophilic bacterium Thermotoga maritima, TmTrpRS, that has recently been structurally characterized. It represents an unprecedented connection among a primordial iron–sulfur cofactor, RNA and protein biosynthesis. Here, a possible role of the [Fe4S4] cluster in tRNA anticodon-loop recognition is investigated by means of density functional theory and comparison with the structure of a human tryptophanyl-tRNA synthetase/tRNA complex. It turns out that a cluster-coordinating cysteine residue, R224, and polar main chain atoms form a characteristic structural motif for recognizing a putative 5′ cytosine or 5′ 2-thiocytosine moiety in the anticodon loop of the tRNA molecule. This motif provides not only affinity but also specificity by creating a structural and energetical penalty for the binding of other bases, such as uracil. PMID:24753428

  1. Origin and Evolution of Glutamyl-prolyl tRNA Synthetase WHEP Domains Reveal Evolutionary Relationships within Holozoa

    PubMed Central

    Ray, Partho Sarothi; Fox, Paul L.

    2014-01-01

    Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering “missing links” in the tree of life. PMID:24968216

  2. Defects in tRNA Modification Associated with Neurological and Developmental Dysfunctions in Caenorhabditis elegans Elongator Mutants

    PubMed Central

    Chen, Changchun; Tuck, Simon; Byström, Anders S.

    2009-01-01

    Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5′methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development. PMID:19593383

  3. Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection.

    PubMed

    Au, Hilda H; Cornilescu, Gabriel; Mouzakis, Kathryn D; Ren, Qian; Burke, Jordan E; Lee, Seonghoon; Butcher, Samuel E; Jan, Eric

    2015-11-24

    The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame. PMID:26554019

  4. Crystal structure of Bacillus subtilis TrmB, the tRNA (m7G46) methyltransferase.

    PubMed

    Zegers, Ingrid; Gigot, Daniel; van Vliet, Françoise; Tricot, Catherine; Aymerich, Stéphane; Bujnicki, Janusz M; Kosinski, Jan; Droogmans, Louis

    2006-01-01

    The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m7G46) methyltransferase, was determined at a resolution of 2.1 A. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-L-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer.

  5. Crystal structure of Bacillus subtilis TrmB, the tRNA (m7G46) methyltransferase

    PubMed Central

    Zegers, Ingrid; Gigot, Daniel; van Vliet, Françoise; Tricot, Catherine; Aymerich, Stéphane; Bujnicki, Janusz M.; Kosinski, Jan; Droogmans, Louis

    2006-01-01

    The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m7G46) methyltransferase, was determined at a resolution of 2.1 Å. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-l-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer. PMID:16600901

  6. The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA.

    PubMed

    Goswami, Srikanta; Adhya, Samit

    2006-07-14

    Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import. PMID:16735512

  7. Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome

    NASA Astrophysics Data System (ADS)

    Adio, Sarah; Senyushkina, Tamara; Peske, Frank; Fischer, Niels; Wintermeyer, Wolfgang; Rodnina, Marina V.

    2015-06-01

    The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

  8. Structure, mechanism, and specificity of a eukaryal tRNA restriction enzyme involved in self-nonself discrimination.

    PubMed

    Chakravarty, Anupam K; Smith, Paul; Jalan, Radhika; Shuman, Stewart

    2014-04-24

    tRNA restriction by anticodon nucleases underlies cellular stress responses and self-nonself discrimination in a wide range of taxa. Anticodon breakage inhibits protein synthesis, which, in turn, results in growth arrest or cell death. The eukaryal ribotoxin PaT secreted by Pichia acaciae inhibits growth of Saccharomyces cerevisiae via cleavage of tRNA(Gln(UUG)). We find that recombinant PaT incises a synthetic tRNA(Gln(UUG)) stem-loop RNA by transesterification at a single site 3' of the wobble uridine, yielding 2',3'-cyclic phosphate and 5'-OH ends. Incision is suppressed by replacement of the wobble nucleobase with adenine or guanine. The crystal structure of PaT reveals a distinctive fold and active site, essential components of which are demonstrated by mutagenesis. Pichia acaciae evades self-toxicity via a distinctive intracellular immunity protein, ImmPaT, which binds PaT and blocks nuclease activity. Our results highlight the evolutionary diversity of tRNA restriction and immunity systems. PMID:24726365

  9. rrnDB: documenting the number of rRNA and tRNA genes in bacteria and archaea.

    PubMed

    Lee, Zarraz May-Ping; Bussema, Carl; Schmidt, Thomas M

    2009-01-01

    A dramatic exception to the general pattern of single-copy genes in bacterial and archaeal genomes is the presence of 1-15 copies of each ribosomal RNA encoding gene. The original version of the Ribosomal RNA Database (rrnDB) cataloged estimates of the number of 16S rRNA-encoding genes; the database now includes the number of genes encoding each of the rRNAs (5S, 16S and 23S), an internally transcribed spacer region, and the number of tRNA genes. The rrnDB has been used largely by microbiologists to predict the relative rate at which microbial populations respond to favorable growth conditions, and to interpret 16S rRNA-based surveys of microbial communities. To expand the functionality of the rrnDB (http://ribosome.mmg.msu.edu/rrndb/index.php), the search engine has been redesigned to allow database searches based on 16S rRNA gene copy number, specific organisms or taxonomic subsets of organisms. The revamped database also computes average gene copy numbers for any collection of entries selected. Curation tools now permit rapid updates, resulting in an expansion of the database to include data for 785 bacterial and 69 archaeal strains. The rrnDB continues to serve as the authoritative, curated source that documents the phylogenetic distribution of rRNA and tRNA genes in microbial genomes.

  10. GidA, a tRNA Modification Enzyme, Contributes to the Growth, and Virulence of Streptococcus suis Serotype 2

    PubMed Central

    Gao, Ting; Tan, Meifang; Liu, Wanquan; Zhang, Chunyan; Zhang, Tengfei; Zheng, Linlin; Zhu, Jiawen; Li, Lu; Zhou, Rui

    2016-01-01

    Glucose-inhibited division protein (GidA), is a tRNA modification enzyme functioning together with MnmE in the addition of a carboxymethylaminomethyl group to position 5 of the anticodon wobble uridine of tRNA. Here, we report a GidA homolog from a Chinese isolate SC-19 of the zoonotic Streptococcus suis serotype 2 (SS2). gidA disruption led to a defective growth, increased capsule thickness, and reduced hemolytic activity. Moreover, the gidA deletion mutant (ΔgidA) displayed reduced mortality and bacterial loads in mice, reduced ability of adhesion to and invasion in epithelial cells, and increased sensitivity to phagocytosis. The iTRAQ analysis identified 372 differentially expressed (182 up- and 190 down-regulated) proteins in ΔgidA and SC-19. Numerous DNA replication, cell division, and virulence associated proteins were downregulated, whereas many capsule synthesis enzymes were upregulated by gidA disruption. This is consistent with the phenotypes of the mutant. Thus, GidA is a translational regulator that plays an important role in the growth, cell division, capsule biosynthesis, and virulence of SS2. Our findings provide new insight into the regulatory function of GidA in bacterial pathogens. PMID:27148493

  11. Autosomal-Recessive Mutations in the tRNA Splicing Endonuclease Subunit TSEN15 Cause Pontocerebellar Hypoplasia and Progressive Microcephaly.

    PubMed

    Breuss, Martin W; Sultan, Tipu; James, Kiely N; Rosti, Rasim O; Scott, Eric; Musaev, Damir; Furia, Bansri; Reis, André; Sticht, Heinrich; Al-Owain, Mohammed; Alkuraya, Fowzan S; Reuter, Miriam S; Abou Jamra, Rami; Trotta, Christopher R; Gleeson, Joseph G

    2016-07-01

    The tRNA splicing endonuclease is a highly evolutionarily conserved protein complex, involved in the cleavage of intron-containing tRNAs. In human it consists of the catalytic subunits TSEN2 and TSEN34, as well as the non-catalytic TSEN54 and TSEN15. Recessive mutations in the corresponding genes of the first three are known to cause pontocerebellar hypoplasia (PCH) types 2A-C, 4, and 5. Here, we report three homozygous TSEN15 variants that cause a milder version of PCH2. The affected individuals showed progressive microcephaly, delayed developmental milestones, intellectual disability, and, in two out of four cases, epilepsy. None, however, displayed the central visual failure seen in PCH case subjects where other subunits of the TSEN are mutated, and only one was affected by the extensive motor defects that are typical in other forms of PCH2. The three amino acid substitutions impacted the protein level of TSEN15 and the stoichiometry of the interacting subunits in different ways, but all resulted in an almost complete loss of in vitro tRNA cleavage activity. Taken together, our results demonstrate that mutations in any known subunit of the TSEN complex can cause PCH and progressive microcephaly, emphasizing the importance of its function during brain development.

  12. Autosomal-Recessive Mutations in the tRNA Splicing Endonuclease Subunit TSEN15 Cause Pontocerebellar Hypoplasia and Progressive Microcephaly.

    PubMed

    Breuss, Martin W; Sultan, Tipu; James, Kiely N; Rosti, Rasim O; Scott, Eric; Musaev, Damir; Furia, Bansri; Reis, André; Sticht, Heinrich; Al-Owain, Mohammed; Alkuraya, Fowzan S; Reuter, Miriam S; Abou Jamra, Rami; Trotta, Christopher R; Gleeson, Joseph G

    2016-07-01

    The tRNA splicing endonuclease is a highly evolutionarily conserved protein complex, involved in the cleavage of intron-containing tRNAs. In human it consists of the catalytic subunits TSEN2 and TSEN34, as well as the non-catalytic TSEN54 and TSEN15. Recessive mutations in the corresponding genes of the first three are known to cause pontocerebellar hypoplasia (PCH) types 2A-C, 4, and 5. Here, we report three homozygous TSEN15 variants that cause a milder version of PCH2. The affected individuals showed progressive microcephaly, delayed developmental milestones, intellectual disability, and, in two out of four cases, epilepsy. None, however, displayed the central visual failure seen in PCH case subjects where other subunits of the TSEN are mutated, and only one was affected by the extensive motor defects that are typical in other forms of PCH2. The three amino acid substitutions impacted the protein level of TSEN15 and the stoichiometry of the interacting subunits in different ways, but all resulted in an almost complete loss of in vitro tRNA cleavage activity. Taken together, our results demonstrate that mutations in any known subunit of the TSEN complex can cause PCH and progressive microcephaly, emphasizing the importance of its function during brain development. PMID:27392077

  13. Live Cell Imaging Using Riboswitch-Spinach tRNA Fusions as Metabolite-Sensing Fluorescent Biosensors.

    PubMed

    Kellenberger, Colleen A; Hallberg, Zachary F; Hammond, Ming C

    2015-01-01

    The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a pro-fluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.

  14. Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection.

    PubMed

    Au, Hilda H; Cornilescu, Gabriel; Mouzakis, Kathryn D; Ren, Qian; Burke, Jordan E; Lee, Seonghoon; Butcher, Samuel E; Jan, Eric

    2015-11-24

    The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.

  15. Nucleocytoplasmic shuttling of tRNAs and implication of the cytosolic Hsp70 system in tRNA import.

    PubMed

    Yoshihisa, Tohru

    2015-01-01

    tRNAs, a class of non-coding RNAs essential for translation, are unique among cytosolic RNA species in that they shuttle between the nucleus and cytoplasm during their life. Although their export from the nucleus has been studied in detail, limited information on import machinery was available. Our group recently reported that Ssa2p, one of major cytosolic Hsp70s in Saccharomyces cerevisiae, acts as a crucial factor for tRNA import upon nutrient starvation. Ssa2p can bind tRNAs and a nucleoporin directly in an ATP-sensitive manner, suggesting that it acts as a nuclear import carrier for tRNAs, like importin-β proteins. In vitro assays revealed that Ssa2p binds tRNA specifically but has preference for loosely folded tRNAs. In this Extra View, these features of Ssa2p as a new import factor is discussed with other recent findings related to nucleocytoplasmic transport of tRNAs reported from other groups. PMID:26280499

  16. Three-Dimensional Algebraic Models of the tRNA Code and 12 Graphs for Representing the Amino Acids.

    PubMed

    José, Marco V; Morgado, Eberto R; Guimarães, Romeu Cardoso; Zamudio, Gabriel S; de Farías, Sávio Torres; Bobadilla, Juan R; Sosa, Daniela

    2014-08-11

    Three-dimensional algebraic models, also called Genetic Hotels, are developed to represent the Standard Genetic Code, the Standard tRNA Code (S-tRNA-C), and the Human tRNA code (H-tRNA-C). New algebraic concepts are introduced to be able to describe these models, to wit, the generalization of the 2n-Klein Group and the concept of a subgroup coset with a tail. We found that the H-tRNA-C displayed broken symmetries in regard to the S-tRNA-C, which is highly symmetric. We also show that there are only 12 ways to represent each of the corresponding phenotypic graphs of amino acids. The averages of statistical centrality measures of the 12 graphs for each of the three codes are carried out and they are statistically compared. The phenotypic graphs of the S-tRNA-C display a common triangular prism of amino acids in 10 out of the 12 graphs, whilst the corresponding graphs for the H-tRNA-C display only two triangular prisms. The graphs exhibit disjoint clusters of amino acids when their polar requirement values are used. We contend that the S-tRNA-C is in a frozen-like state, whereas the H-tRNA-C may be in an evolving state.

  17. tRNA tKUUU, tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding

    PubMed Central

    Rezgui, Vanessa Anissa Nathalie; Tyagi, Kshitiz; Ranjan, Namit; Konevega, Andrey L.; Mittelstaet, Joerg; Rodnina, Marina V.; Peter, Matthias; Pedrioli, Patrick G. A.

    2013-01-01

    tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic (tKUUU), (tQUUG), and (tEUUC). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1- and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tKUUU to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content. PMID:23836657

  18. The Cm56 tRNA modification in archaea is catalyzed either by a specific 2′-O-methylase, or a C/D sRNP

    PubMed Central

    RENALIER, MARIE-HÉLÈNE; JOSEPH, NICOLE; GASPIN, CHRISTINE; THEBAULT, PATRICIA; MOUGIN, ANNIE

    2005-01-01

    We identified the first archaeal tRNA ribose 2′-O-methylase, aTrm56, belonging to the Cluster of Orthologous Groups (COG) 1303 that contains archaeal genes only. The corresponding protein exhibits a SPOUT S-adenosylmethionine (AdoMet)-dependent methyltransferase domain found in bacterial and yeast G18 tRNA 2′-O-methylases (SpoU, Trm3). We cloned the Pyrococcus abyssi PAB1040 gene belonging to this COG, expressed and purified the corresponding protein, and showed that in vitro, it specifically catalyzes the AdoMet-dependent 2′-O-ribose methylation of C at position 56 in tRNA transcripts. This tRNA methylation is present only in archaea, and the gene for this enzyme is present in all the archaeal genomes sequenced up to now, except in the crenarchaeon Pyrobaculum aerophilum. In this archaea, the C56 2′-O-methylation is provided by a C/D sRNP. Our work is the first demonstration that, within the same kingdom, two different mechanisms are used to modify the same nucleoside in tRNAs. PMID:15987815

  19. Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA – A Possible Intermediate in Its Selenation?

    PubMed Central

    Jäger, Gunilla; Chen, Peng; Björk, Glenn R.

    2016-01-01

    The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm5s2U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U) in tRNA specific for Gln. The sulfur of (c)mnm5s2U may be exchanged by selenium (Se)–a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C10H17-fragment) attached to the s2 group of mnm5s2U and of cmnm5s2U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm5ges2U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH+ also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c)mnm5s2U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction

  20. Clues to tRNA Evolution from the Distribution of Class II tRNAs and Serine Codons in the Genetic Code.

    PubMed

    Bernhardt, Harold S

    2016-01-01

    We have previously proposed that tRNA(Gly) was the first tRNA and glycine was the first amino acid incorporated into the genetic code. The next two amino acids incorporated would have been the other two small hydrophilic amino acids serine and aspartic acid, which occurred through the duplication of the tRNA(Gly) sequence, followed by mutation of its anticodon by single C to U transition mutations, possibly through spontaneous deamination. Interestingly, however, tRNA(Ser) has a different structure than most other tRNAs, possessing a long variable arm; because of this tRNA(Ser) is classified as a class II tRNA. Also, serine codons are found not only in the bottom right-hand corner of the genetic code table next to those for glycine and aspartic acid, but also in the top row of the table, next to those for two of the most hydrophobic amino acids, leucine and phenylalanine. In the following, I propose that the class II tRNA structure of tRNA(Ser) and the arrangement of serine codons in the genetic code provide clues to the early evolution of tRNA and the genetic code. In addition, I address Di Giulio's recent criticism of our proposal that tRNA(Gly) was the first tRNA, and discuss how early peptides produced from a restricted amino acid alphabet of glycine, serine and aspartic acid might have possessed proteolytic activity, which is possibly important for the early recycling of amino acid monomers. PMID:26927183

  1. Clues to tRNA Evolution from the Distribution of Class II tRNAs and Serine Codons in the Genetic Code

    PubMed Central

    Bernhardt, Harold S.

    2016-01-01

    We have previously proposed that tRNAGly was the first tRNA and glycine was the first amino acid incorporated into the genetic code. The next two amino acids incorporated would have been the other two small hydrophilic amino acids serine and aspartic acid, which occurred through the duplication of the tRNAGly sequence, followed by mutation of its anticodon by single C to U transition mutations, possibly through spontaneous deamination. Interestingly, however, tRNASer has a different structure than most other tRNAs, possessing a long variable arm; because of this tRNASer is classified as a class II tRNA. Also, serine codons are found not only in the bottom right-hand corner of the genetic code table next to those for glycine and aspartic acid, but also in the top row of the table, next to those for two of the most hydrophobic amino acids, leucine and phenylalanine. In the following, I propose that the class II tRNA structure of tRNASer and the arrangement of serine codons in the genetic code provide clues to the early evolution of tRNA and the genetic code. In addition, I address Di Giulio’s recent criticism of our proposal that tRNAGly was the first tRNA, and discuss how early peptides produced from a restricted amino acid alphabet of glycine, serine and aspartic acid might have possessed proteolytic activity, which is possibly important for the early recycling of amino acid monomers. PMID:26927183

  2. Diversity of the biosynthesis pathway for threonylcarbamoyladenosine (t6A), a universal modification of tRNA

    PubMed Central

    Thiaville, Patrick C; Iwata-Reuyl, Dirk; de Crécy-Lagard, Valérie

    2014-01-01

    The tRNA modification field has a rich literature covering biochemical analysis going back more than 40 years, but many of the corresponding genes were only identified in the last decade. In recent years, comparative genomic-driven analysis has allowed for the identification of the genes and subsequent characterization of the enzymes responsible for N6-threonylcarbamoyladenosine (t6A). This universal modification, located in the anticodon stem-loop at position 37 adjacent to the anticodon of tRNAs, is found in nearly all tRNAs that decode ANN codons. The t6A biosynthesis enzymes and synthesis pathways have now been identified, revealing both a core set of enzymes and kingdom-specific variations. This review focuses on the elucidation of the pathway, diversity of the synthesis genes, and proposes a new nomenclature for t6A synthesis enzymes. PMID:25629598

  3. Crystallization and preliminary crystallographic analysis of tRNA (m(7)G46) methyltransferase from Escherichia coli.

    PubMed

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun; Niu, Liwen

    2008-08-01

    Transfer RNA (tRNA) (m(7)G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-L-methionine (SAM) as the methyl-group donor to catalyze the formation of N(7)-methylguanosine (m(7)G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His(6) tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P2(1).

  4. Crystallization and preliminary crystallographic analysis of tRNA (m7G46) methyltransferase from Escherichia coli

    PubMed Central

    Liu, Qi; Gao, Yang; Yang, Weili; Zhou, Huihao; Gao, Yongxiang; Zhang, Xiao; Teng, Maikun; Niu, Liwen

    2008-01-01

    Transfer RNA (tRNA) (m7G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N 7-­methylguanosine (m7G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P21. PMID:18678947

  5. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions.

    PubMed

    Long, Yicheng; Jackman, Jane E

    2015-07-22

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from Acanthamoeba castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing.

  6. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions

    PubMed Central

    Long, Yicheng; Jackman, Jane E.

    2015-01-01

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from A. castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing. PMID:26143376

  7. Overproduction, crystallization and preliminary X-ray crystallographic analysis of Escherichia coli tRNA N 6-threonylcarbamoyladenosine dehydratase

    PubMed Central

    Kim, Sunmin; Kim, Keon Young; Park, Jeong Kuk; Lee, Byung Il; Kim, Yun-Gon; Park, SangYoun

    2014-01-01

    Escherichia coli tRNA N 6-threonylcarbamoyladenosine dehydratase (TcdA), previously called CsdL or YgdL, was overproduced and purified from E. coli and crystallized using polyethylene glycol 3350 as a crystallizing agent. X-ray diffraction data were collected to 2.70 Å resolution under cryoconditions using synchrotron X-rays. The crystals belonged to space group P21, with unit-cell parameters a = 65.4, b = 96.8, c = 83.3 Å, β = 111.7°. According to the Matthews coefficient, the asymmetric unit may contain up to four subunits of the monomeric protein, with a crystal volume per protein mass (V M) of 2.12 Å3 Da−1 and 42.1% solvent content. PMID:25372820

  8. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA.

    PubMed Central

    Barat, C; Lullien, V; Schatz, O; Keith, G; Nugeyre, M T; Grüninger-Leitch, F; Barré-Sinoussi, F; LeGrice, S F; Darlix, J L

    1989-01-01

    The virion cores of the replication competent type 1 human immunodeficiency virus (HIV-1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV-1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV-1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100-fold molar excess of other tRNAs. Cross-linking analysis of this interaction locates the contact site to a region within the heavily modified anti-codon domain of tRNALys,3. Images PMID:2479543

  9. Preliminary X-ray crystallographic analysis of tRNA pseudouridine 55 synthase from the thermophilic eubacterium Thermotoga maritima.

    PubMed

    Wouters, Johan; Tricot, Catherine; Durbecq, Virginie; Roovers, Martine; Stalon, Victor; Droogmans, Louis

    2003-01-01

    Thermotoga maritima TruB, an enzyme responsible for the formation of pseudouridine in tRNA, has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mM citrate pH 3.5, 200 mM Li(2)SO(4), 20% glycerol, 13% PEG 8000. Crystals display orthorhombic symmetry, with unit-cell parameters a = 47.39, b = 83.88, c = 98.72 A, and diffract to 2.0 A resolution using synchrotron radiation. A solution was obtained by molecular replacement using part of the recently published crystal structure of Escherichia coli TruB bound to a synthetic RNA.

  10. Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

    SciTech Connect

    Evdokimov, Artem G.; Mekel, Marlene; Hutchings, Kim; Narasimhan, Lakshmi; Holler, Tod; McGrath, Teresa; Beattie, Bryan; Fauman, Eric; Yan, Chunhong; Heaslet, Holly; Walter, Richard; Finzel, Barry; Ohren, Jeffrey; McConnell, Patrick; Braden, Timothy; Sun, Fang; Spessard, Cindy; Banotai, Craig; Al-Kassim, Loola; Ma, Weijun; Wengender, Paul; Kole, Denis; Garceau, Norman; Toogood, Peter; Liu, Jia

    2008-07-08

    In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality -- they only diffracted X-rays to 3--5 {angstrom} resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 {angstrom} or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

  11. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo

    PubMed Central

    Rijal, Keshab; Maraia, Richard J.

    2016-01-01

    The ability of RNA polymerase (RNAP) III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC); they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT) mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease. PMID:27518095

  12. Global translational impacts of the loss of the tRNA modification t6A in yeast

    PubMed Central

    Thiaville, Patrick C.; Legendre, Rachel; Rojas-Benítez, Diego; Baudin-Baillieu, Agnès; Hatin, Isabelle; Chalancon, Guilhem; Glavic, Alvaro; Namy, Olivier; de Crécy-Lagard, Valérie

    2016-01-01

    The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt) and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs) were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes. PMID:26798630

  13. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis. PMID:27402763

  14. The MiaA tRNA Modification Enzyme Is Necessary for Robust RpoS Expression in Escherichia coli

    PubMed Central

    Gottesman, Susan

    2014-01-01

    The stationary phase/general stress response sigma factor RpoS (σS) is necessary for adaptation and restoration of homeostasis in stationary phase. As a physiological consequence, its levels are tightly regulated at least at two levels. Multiple small regulatory RNA molecules modulate its translation, in a manner that is dependent on the RNA chaperone Hfq and the rpoS 5′ untranslated region. ClpXP and the RssB adaptor protein degrade RpoS, unless it is protected by an anti-adaptor. We here find that, in addition to these posttranscriptional levels of regulation, tRNA modification also affects the steady-state levels of RpoS. We screened mutants of several RNA modification enzymes for an effect on RpoS expression and identified the miaA gene, encoding a tRNA isopentenyltransferase, as necessary for full expression of both an rpoS750-lacZ translational fusion and the RpoS protein. This effect is independent of rpoS, the regulatory RNAs, and RpoS degradation. RpoD steady-state levels were not significantly different in the absence of MiaA, suggesting that this is an RpoS-specific effect. The rpoS coding sequence is significantly enriched for leu codons that use MiaA-modified tRNAs, compared to rpoD and many other genes. Dependence on MiaA may therefore provide yet another way for RpoS levels to respond to growth conditions. PMID:24296670

  15. The C-terminal region of thermophilic tRNA (m7G46) methyltransferase (TrmB) stabilizes the dimer structure and enhances fidelity of methylation.

    PubMed

    Tomikawa, Chie; Ochi, Anna; Hori, Hiroyuki

    2008-05-15

    Transfer RNA (m(7)G46) methyltransferase catalyzes methyl-transfer from S-adenosyl-L-methionine to N(7) atom of the semi-conserved G46 base in tRNA. Aquifex aeolicus is a hyper thermophilic eubacterium that grows at close to 95 degrees C. A. aeolicus tRNA (m(7)G46) methyltransferase [TrmB] has an elongated C-terminal region as compared with mesophilic counterparts. In this study, the authors focused on the functions of this C-terminal region. Analytic gel filtration chromatography and amino acid sequencing reveled that the start point (Glu202) of the C-terminal region is often cleaved by proteases during purification steps and the C-terminal region tightly binds to another subunit even in the presence of 6M urea. Because the C-terminal region contains abundant basic amino acid residues, the authors assumed that some of these residues might be involved in tRNA binding. To address this idea, the authors prepared eight alanine substitution mutant proteins. However, measurements of initial velocities of these mutant proteins suggested that the basic amino acid residues in the C-terminal region are not involved in tRNA binding. The authors investigated effects of the deletion of the C-terminal region. Deletion mutant protein of the C-terminal region (the core protein) was precipitated by incubation at 85 degrees C, while the wild type protein was soluble at that temperature, demonstrating that the C-terminal region contributes to the protein stability at high temperatures. The core protein had a methyl-transfer activity to yeast tRNA(Phe) transcript. Furthermore, the core protein slowly methylated tRNA transcripts, which did not contain G46 base. Moreover, the modified base was identified as m(7)G by two-dimensional thin layer chromatography. Thus, the deletion of the C-terminal region causes nonspecific methylation of N(7) atom of guanine base(s) in tRNA transcripts.

  16. A unique tRNA gene family and a novel, highly expressed ORF in the mitochondrial genome of the silver-lip pearl oyster, Pinctada maxima (Bivalvia: Pteriidae).

    PubMed

    Wu, Xiangyun; Li, Xiaoling; Li, Lu; Yu, Ziniu

    2012-11-15

    Characteristics of mitochondrial (mt) DNA such as gene content and arrangement, as well as mt tRNA secondary structure, are frequently used in comparative genomic analyses because they provide valuable phylogenetic information. However, most analyses do not characterize the relationship of tRNA genes from the same mt genome and, in some cases, analyses overlook possible novel open reading frames (ORFs) when the 13 expected protein-coding genes are already annotated. In this study, we describe the sequence and characterization of the complete mt genome of the silver-lip pearl oyster, Pinctada maxima. The 16,994-bp mt genome contains the same 13 protein-coding genes (PCGs) and two ribosomal RNA genes typical of metazoans. The gene arrangement, however, is completely distinct from that of all other available bivalve mt genomes, and a unique tRNA gene family is observed in this genome. The unique tRNA gene family includes two trnS(-AGY) and trnQ genes, a trnM isomerism, but it lacks trnS(-CUN). We also report the first clear evidence of alloacceptor tRNA gene recruitment (trnP→trnS(-AGY)) in mollusks. In addition, a novel ORF (orfUR1) expressed at high levels is present in the mt genome of this pearl oyster. This gene contains a conserved domain, "Oxidored_q1_N", which is a member of Complex I and thus may play an important role in key biological functions. Because orfUR1 has a very similar nucleotide composition and codon bias to that of other genes in this genome, we hypothesize that this gene may have been moved to the mt genome via gene transfer from the nuclear genome at an early stage of speciation of P. maxima, or it may have evolved as a result of gene duplication, followed by rapid sequence divergence. Lastly, a 319-bp region was identified as the possible control region (CR) even though it does not correspond to the longest non-coding region in the genome. Unlike other studies of mt genomes, this study compares the evolutionary patterns of all available

  17. Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells

    PubMed Central

    Köhrer, Caroline; Sullivan, Eric L.; RajBhandary, Uttam L.

    2004-01-01

    We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells. PMID:15576346

  18. A platform for discovery and quantification of modified ribonucleosides in RNA: Application to stress-induced reprogramming of tRNA modifications

    PubMed Central

    Cai, Weiling Maggie; Chionh, Yok Hian; Hia, Fabian; Gu, Chen; Kellner, Stefanie; McBee, Megan E.; Ng, Chee Sheng; Pang, Yan Ling Joy; Prestwich, Erin G.; Lim, Kok Seong; Babu, I. Ramesh; Begley, Thomas J.; Dedon, Peter C.

    2016-01-01

    Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. The chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: 1) RNA isolation, 2) RNA purification, 3) RNA hydrolysis to individual ribonucleosides, 4) chromatographic resolution of ribonucleosides, 5) identification of the full set of modified ribonucleosides, 6) mass spectrometric quantification of ribonucleosides, 6) interrogation of ribonucleoside datasets, and 7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications. PMID:26253965

  19. MTO1 mediates tissue specificity of OXPHOS defects via tRNA modification and translation optimization, which can be bypassed by dietary intervention.

    PubMed

    Tischner, Christin; Hofer, Annette; Wulff, Veronika; Stepek, Joanna; Dumitru, Iulia; Becker, Lore; Haack, Tobias; Kremer, Laura; Datta, Alexandre N; Sperl, Wolfgang; Floss, Thomas; Wurst, Wolfgang; Chrzanowska-Lightowlers, Zofia; De Angelis, Martin Hrabe; Klopstock, Thomas; Prokisch, Holger; Wenz, Tina

    2015-04-15

    Mitochondrial diseases often exhibit tissue-specific pathologies, but this phenomenon is poorly understood. Here we present regulation of mitochondrial translation by the Mitochondrial Translation Optimization Factor 1, MTO1, as a novel player in this scenario. We demonstrate that MTO1 mediates tRNA modification and controls mitochondrial translation rate in a highly tissue-specific manner associated with tissue-specific OXPHOS defects. Activation of mitochondrial proteases, aberrant translation products, as well as defects in OXPHOS complex assembly observed in MTO1 deficient mice further imply that MTO1 impacts translation fidelity. In our mouse model, MTO1-related OXPHOS deficiency can be bypassed by feeding a ketogenic diet. This therapeutic intervention is independent of the MTO1-mediated tRNA modification and involves balancing of mitochondrial and cellular secondary stress responses. Our results thereby establish mammalian MTO1 as a novel factor in the tissue-specific regulation of OXPHOS and fine tuning of mitochondrial translation accuracy.

  20. Resistivity of Carbon-Carbon Composites Halved

    NASA Technical Reports Server (NTRS)

    Gaier, James R.

    2004-01-01

    Carbon-carbon composites have become the material of choice for applications requiring strength and stiffness at very high temperatures (above 2000 C). These composites comprise carbon or graphite fibers embedded in a carbonized or graphitized matrix. In some applications, such as shielding sensitive electronics in very high temperature environments, the performance of these materials would be improved by lowering their electrical resistivity. One method to lower the resistivity of the composites is to lower the resistivity of the graphite fibers, and a proven method to accomplish that is intercalation. Intercalation is the insertion of guest atoms or molecules into a host lattice. In this study the host fibers were highly graphitic pitch-based graphite fibers, or vapor-grown carbon fibers (VGCF), and the intercalate was bromine. Intercalation compounds of graphite are generally thought of as being only metastable, but it has been shown that the residual bromine graphite fiber intercalation compound is remarkably stable, resisting decomposition even at temperatures at least as high as 1000 C. The focus of this work was to fabricate composite preforms, determine whether the fibers they were made from were still intercalated with bromine after processing, and determine the effect on composite resistivity. It was not expected that the resistivity would be lowered as dramatically as with graphite polymer composites because the matrix itself would be much more conductive, but it was hoped that the gains would be substantial enough to warrant its use in high-performance applications. In a collaborative effort supporting a Space Act Agreement between the NASA Glenn Research Center and Applied Sciences, Inc. (Cedarville, OH), laminar preforms were fabricated with pristine and bromine-intercalated pitch-based fibers (P100 and P100-Br) and VGCF (Pyro I and Pyro I-Br). The green preforms were carbonized at 1000 C and then heat treated to 3000 C. To determine whether the fibers in the samples were still intercalated after composite fabrication, they were subjected to X-ray diffraction. The composites containing intercalated graphite fibers showed much higher background scatter than that of pristine fibers, indicating the presence of bromine in the samples. More importantly, faint features indicative of intercalation were visible in the diffraction pattern, showing that the fibers were still intercalated.

  1. A methods review on use of nonsense suppression to study 3′ end formation and other aspects of tRNA biogenesis

    PubMed Central

    Rijal, Keshab; Maraia, Richard J.; Arimbasseri, Aneeshkumar G.

    2014-01-01

    Suppressor tRNAs bear anticodon mutations that allow them to decode premature stop codons in metabolic marker gene mRNAs, that can be used as in vivo reporters of functional tRNA biogenesis. Here, we review key components of a suppressor tRNA system specific to S. pombe and its adaptations for use to study specific steps in tRNA biogenesis. Eukaryotic tRNA biogenesis begins with transcription initiation by RNA polymerase (pol) III. The nascent pre-tRNAs must undergo folding, 5′ and 3′ processing to remove the leader and trailer, nuclear export, and splicing if applicable, while multiple complex chemical modifications occur throughout the process. We review evidence that precursor-tRNA processing begins with transcription termination at the oligo(T) terminator element, which forms a 3′ oligo(U) tract on the nascent RNA, a sequence-specific binding site for the RNA chaperone, La protein. The processing pathway bifurcates depending on a poorly understood property of pol III termination that determines the 3′ oligo(U) length and therefore the affinity for La. We thus review the pol III termination process and the factors involved including advances using gene-specific random mutagenesis by dNTP analogs that identify key residues important for transcription termination in certain pol III subunits. The review ends with a ‘technical approaches’ section that includes a parts lists of suppressor-tRNA alleles, strains and plasmids, and graphic examples of its diverse uses. PMID:25447915

  2. Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance.

    PubMed

    Kinghorn, Seonag M; O'Byrne, Conor P; Booth, Ian R; Stansfield, Ian

    2002-11-01

    The truB gene of Escherichia coli encodes the pseudouridine-55 (psi55) synthase and is responsible for modifying all tRNA molecules in the cell at the U55 position. A truB null mutant grew normally on all growth media tested, but exhibited a competitive disadvantage in extended co-culture with its wild-type progenitor. The mutant phenotype could be complemented by both the cloned truB gene and by a D48C, catalytically inactive allele of truB. The truB mutant also exhibited a defect in survival of rapid transfer from 37 to 50 degrees C. This mutant phenotype could be complemented by the cloned truB gene but not by a D48C, catalytically inactive allele of truB. The temperature sensitivity of truB mutants could be enhanced by combination with a mutation in the trmA gene, encoding an m(5)U-methyltransferase, modifying the universal U54 tRNA nucleoside, but not by mutations in trmH, encoding the enzyme catalysing the formation of Gm18. The truB mutant proteome contained altered levels of intermediates involved in biogenesis of the outer-membrane proteins OmpA and OmpX. The truB mutation also reduced the basal expression from two sigma(E) promoters, degP and rpoHP3. Three novel aspects to the phenotype of truB mutants were identified. Importantly the data support the hypothesis that TruB-effected psi55 modification of tRNA is not essential, but contributes to thermal stress tolerance in E. coli, possibly by optimizing the stability of the tRNA population at high temperatures.

  3. Defective i6A37 modification of mitochondrial and cytosolic tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA.

    PubMed

    Yarham, John W; Lamichhane, Tek N; Pyle, Angela; Mattijssen, Sandy; Baruffini, Enrico; Bruni, Francesco; Donnini, Claudia; Vassilev, Alex; He, Langping; Blakely, Emma L; Griffin, Helen; Santibanez-Koref, Mauro; Bindoff, Laurence A; Ferrero, Ileana; Chinnery, Patrick F; McFarland, Robert; Maraia, Richard J; Taylor, Robert W

    2014-06-01

    Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.

  4. Defective i6A37 Modification of Mitochondrial and Cytosolic tRNAs Results from Pathogenic Mutations in TRIT1 and Its Substrate tRNA

    PubMed Central

    Pyle, Angela; Mattijssen, Sandy; Baruffini, Enrico; Bruni, Francesco; Donnini, Claudia; Vassilev, Alex; He, Langping; Blakely, Emma L.; Griffin, Helen; Santibanez-Koref, Mauro; Bindoff, Laurence A.; Ferrero, Ileana; Chinnery, Patrick F.; McFarland, Robert; Maraia, Richard J.; Taylor, Robert W.

    2014-01-01

    Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs. PMID:24901367

  5. The antimicrobial natural product chuangxinmycin and some synthetic analogues are potent and selective inhibitors of bacterial tryptophanyl tRNA synthetase.

    PubMed

    Brown, Murray J; Carter, Paul S; Fenwick, Ashley S; Fosberry, Andrew P; Hamprecht, Dieter W; Hibbs, Martin J; Jarvest, Richard L; Mensah, Lucy; Milner, Peter H; O'Hanlon, Peter J; Pope, Andrew J; Richardson, Christine M; West, Andrew; Witty, David R

    2002-11-01

    The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.

  6. Aminoacyl-tRNA synthetases are multivalent suppressors of defects due to human equivalent mutations in yeast mt tRNA genes.

    PubMed

    Montanari, Arianna; De Luca, Cristina; Frontali, Laura; Francisci, Silvia

    2010-09-01

    The use of the yeast model for the study of the molecular and cellular effects of the pathogenic base substitutions in human mitochondrial tRNA genes has recently been validated by the finding that the suppressing factors identified in yeast (the mitochondrial protein elongation factor EF-Tu and the cognate aminoacyl-tRNA synthetase) have suppressing activities also in human cells. In this paper we report a detailed analysis of the cross-suppressing activities of valyl- and leucyl-tRNA synthetases on different tRNA mutants. Glycerol growth, respiration, Northern analysis consistently show that similar suppressing effects can be obtained by these two yeast synthetases and by the orthologous human enzymes. As a whole the present data indicate that the suppression by mt aa-RS is probably not related to the enzyme activities per se, and may be due to a stabilizing chaperon-like effect of the synthetase molecules on the tRNA structure altered by the mutations.

  7. Perspectives and Insights into the Competition for Aminoacyl-tRNAs between the Translational Machinery and for tRNA Dependent Non-Ribosomal Peptide Bond Formation.

    PubMed

    Fung, Angela W S; Payoe, Roshani; Fahlman, Richard P

    2015-01-01

    Aminoacyl-tRNA protein transferases catalyze the transfer of amino acids from aminoacyl-tRNAs to polypeptide substrates. Different forms of these enzymes are found in the different kingdoms of life and have been identified to be central to a wide variety of cellular processes. L/F-transferase is the sole member of this class of enzyme found in Escherichia coli and catalyzes the transfer of leucine to the N-termini of proteins which result in the targeted degradation of the modified protein. Recent investigations on the tRNA specificity of L/F-transferase have revealed the unique recognition nucleotides for a preferred Leu-tRNA(Leu) isoacceptor substrate. In addition to discussing this tRNA selectivity by L/F-transferase, we present and discuss a hypothesis and its implications regarding the apparent competition for this aminoacyl-tRNA between L/F-transferase and the translational machinery. Our discussion reveals a hypothetical involvement of the bacterial stringent response that occurs upon amino acid limitation as a potential cellular event that may reduce this competition and provide the opportunity for L/F-transferase to readily increase its access to the pool of aminoacylated tRNA substrates.

  8. Non-Conserved Residues in Clostridium acetobutylicum tRNAAla Contribute to tRNA Tuning for Efficient Antitermination of the alaS T Box Riboswitch

    PubMed Central

    Liu, Liang-Chun; Grundy, Frank J.; Henkin, Tina M.

    2015-01-01

    The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient antitermination. PMID:26426057

  9. Hetero subunit interaction and RNA recognition of yeast tRNA (m7G46) methyltransferase synthesized in a wheat germ cell-free translation system.

    PubMed

    Muneyoshi, Yuki; Matsumoto, Keisuke; Tomikawa, Chie; Toyooka, Takashi; Ochi, Anna; Masaoka, Takashi; Endo, Yaeta; Hori, Hiroyuki

    2007-01-01

    Yeast tRNA (m(7)G46) methyltransferase contains two protein subunits (Trm8 and Trm82). The enzyme catalyzes a methyl-transfer from S-adenosyl-L-methionine to the N(7) atom of guanine at position 46 in tRNA. We deviced synthesis of active Trm8-Trm82 heterodimer in a wheat germ cell-free translation system. When Trm8 or Trm82 mRNA were used for a synthesis, Trm8 or Trm82 protein could be synthesized. Upon mixing the synthesized Trm8 and Trm82 proteins, no active Trm8-Trm82 heterodimer was produced. Active Trm8-Trm82 heterodimer was only synthesized under conditions, in which both Trm8 and Trm82 mRNAs were co-translated. To address the RNA recognition mechanism of the Trm8-Trm82 complex, we investigated methyl acceptance activities of eight truncated yeast tRNA(Phe) transcripts. In this meeting, we demonstrate that yeast Trm8-Trm82 has stricter recognition requirements for the tRNA molecule as compared to the bacterial enzyme, TrmB.

  10. Crystal structure of the two-subunit tRNA m(1)A58 methyltransferase TRM6-TRM61 from Saccharomyces cerevisiae.

    PubMed

    Wang, Mingxing; Zhu, Yuwei; Wang, Chongyuan; Fan, Xiaojiao; Jiang, Xuguang; Ebrahimi, Mohammad; Qiao, Zhi; Niu, Liwen; Teng, Maikun; Li, Xu

    2016-01-01

    The N(1) methylation of adenine at position 58 (m(1)A58) of tRNA is an important post-transcriptional modification, which is vital for maintaining the stability of the initiator methionine tRNAi(Met). In eukaryotes, this modification is performed by the TRM6-TRM61 holoenzyme. To understand the molecular mechanism that underlies the cooperation of TRM6 and TRM61 in the methyl transfer reaction, we determined the crystal structure of TRM6-TRM61 holoenzyme from Saccharomyces cerevisiae in the presence and absence of its methyl donor S-Adenosyl-L-methionine (SAM). In the structures, two TRM6-TRM61 heterodimers assemble as a heterotetramer. Both TRM6 and TRM61 subunits comprise an N-terminal β-barrel domain linked to a C-terminal Rossmann-fold domain. TRM61 functions as the catalytic subunit, containing a methyl donor (SAM) binding pocket. TRM6 diverges from TRM61, lacking the conserved motifs used for binding SAM. However, TRM6 cooperates with TRM61 forming an L-shaped tRNA binding regions. Collectively, our results provide a structural basis for better understanding the m(1)A58 modification of tRNA occurred in Saccharomyces cerevisiae. PMID:27582183

  11. Phenotypic suppression of DNA gyrase deficiencies by a deletion lowering the gene dosage of a major tRNA in Salmonella typhimurium.

    PubMed Central

    Blanc-Potard, A B; Bossi, L

    1994-01-01

    One of the pleiotropic phenotypes of mutations affecting DNA gyrase activity in Salmonella typhimurium is the constitutive deattenuation of the histidine operon. In the present work, we isolated and characterized a suppressor mutation which restores his attenuation in the presence of a defective gyrase. Such a suppressor, initially named sgdA1 (for suppressor gyrase deficiency), was found to correct additional phenotypes associated with defective gyrase function. These include the aberrant nucleoid partitioning of a gyrB mutant and the conditional lethality of a gyrA mutation. Furthermore, the sgdA1 mutation was found to confer low-level resistance to nalidixic acid. The last phenotype permitted isolation of a number of additional sgdA mutants. Genetic analysis established the recessive character of these alleles as well as the position of the sgdA locus at 57 U on the Salmonella genetic map. All of the sgdA mutants result from the same molecular event: a deletion removing three of the four tandemly repeated copies of argV, the gene which specifies tRNA(2Arg), the major arginine isoacceptor tRNA. These findings, combined with the observation of some Sgd-like phenotypes in a tRNA modification mutant (hisT mutant), lead us to propose that protein synthesis contributes, directly or indirectly, to the pathology of gyrase alterations in growing bacteria. We discuss plausible mechanisms which may be responsible for these effects. Images PMID:7512550

  12. Perspectives and Insights into the Competition for Aminoacyl-tRNAs between the Translational Machinery and for tRNA Dependent Non-Ribosomal Peptide Bond Formation

    PubMed Central

    Fung, Angela W. S.; Payoe, Roshani; Fahlman, Richard P.

    2015-01-01

    Aminoacyl-tRNA protein transferases catalyze the transfer of amino acids from aminoacyl-tRNAs to polypeptide substrates. Different forms of these enzymes are found in the different kingdoms of life and have been identified to be central to a wide variety of cellular processes. L/F-transferase is the sole member of this class of enzyme found in Escherichia coli and catalyzes the transfer of leucine to the N-termini of proteins which result in the targeted degradation of the modified protein. Recent investigations on the tRNA specificity of L/F-transferase have revealed the unique recognition nucleotides for a preferred Leu-tRNALeu isoacceptor substrate. In addition to discussing this tRNA selectivity by L/F-transferase, we present and discuss a hypothesis and its implications regarding the apparent competition for this aminoacyl-tRNA between L/F-transferase and the translational machinery. Our discussion reveals a hypothetical involvement of the bacterial stringent response that occurs upon amino acid limitation as a potential cellular event that may reduce this competition and provide the opportunity for L/F-transferase to readily increase its access to the pool of aminoacylated tRNA substrates. PMID:26729173

  13. Arginine aminoacylation identity is context-dependent and ensured by alternate recognition sets in the anticodon loop of accepting tRNA transcripts.

    PubMed Central

    Sissler, M; Giegé, R; Florentz, C

    1996-01-01

    Yeast arginyl-tRNA synthetase recognizes the non-modified wild-type transcripts derived from both yeast tRNA(Arg) and tRNA(Asp) with equal efficiency. It discriminates its cognate natural substrate, tRNA(Arg), from non-cognate tRNA(Asp) by a negative discrimination mechanism whereby a single methyl group acts as an anti-determinant. Considering these facts, recognition elements responsible for specific arginylation in yeast have been searched by studying the in vitro arginylation properties of a series of transcripts derived from yeast tRNA(Asp), considered as an arginine isoacceptor tRNA. In parallel, experiments on similar tRNA(Arg) transcripts were performed. Unexpectedly, in the tRNA(Arg) context, arginylation is basically linked to the presence of residue C35, whereas in the tRNA(Asp) context, it is deeply related to that of C36 and G37 but is insensitive to the nucleotide at position 35. Each of these nucleotides present in one host, is absent in the other host tRNA. Thus, arginine identity is dependent on two different specific recognition sets according to the tRNA framework investigated. Images PMID:8890180

  14. Crystal structure of the two-subunit tRNA m1A58 methyltransferase TRM6-TRM61 from Saccharomyces cerevisiae

    PubMed Central

    Wang, Mingxing; Zhu, Yuwei; Wang, Chongyuan; Fan, Xiaojiao; Jiang, Xuguang; Ebrahimi, Mohammad; Qiao, Zhi; Niu, Liwen; Teng, Maikun; Li, Xu

    2016-01-01

    The N1 methylation of adenine at position 58 (m1A58) of tRNA is an important post-transcriptional modification, which is vital for maintaining the stability of the initiator methionine tRNAiMet. In eukaryotes, this modification is performed by the TRM6-TRM61 holoenzyme. To understand the molecular mechanism that underlies the cooperation of TRM6 and TRM61 in the methyl transfer reaction, we determined the crystal structure of TRM6-TRM61 holoenzyme from Saccharomyces cerevisiae in the presence and absence of its methyl donor S-Adenosyl-L-methionine (SAM). In the structures, two TRM6-TRM61 heterodimers assemble as a heterotetramer. Both TRM6 and TRM61 subunits comprise an N-terminal β-barrel domain linked to a C-terminal Rossmann-fold domain. TRM61 functions as the catalytic subunit, containing a methyl donor (SAM) binding pocket. TRM6 diverges from TRM61, lacking the conserved motifs used for binding SAM. However, TRM6 cooperates with TRM61 forming an L-shaped tRNA binding regions. Collectively, our results provide a structural basis for better understanding the m1A58 modification of tRNA occurred in Saccharomyces cerevisiae. PMID:27582183

  15. Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code.

    PubMed

    Rozov, Alexey; Demeshkina, Natalia; Khusainov, Iskander; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

    2016-01-01

    Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNA(Lys)(UUU) with hypermodified 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine-pyrimidine mismatches. We show that mnm(5)s(2)U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism. PMID:26791911

  16. Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates

    PubMed Central

    Bar-Yaacov, Dan; Frumkin, Idan; Yashiro, Yuka; Schlesinger, Orr; Bieri, Philipp; Greber, Basil; Ban, Nenad; Zarivach, Raz; Alfonta, Lital; Pilpel, Yitzhak; Suzuki, Tsutomu; Mishmar, Dan

    2016-01-01

    The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function. PMID:27631568

  17. Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code

    PubMed Central

    Rozov, Alexey; Demeshkina, Natalia; Khusainov, Iskander; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

    2016-01-01

    Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNALysUUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine–pyrimidine mismatches. We show that mnm5s2U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism. PMID:26791911

  18. Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

    PubMed Central

    Niehues, Sven; Bussmann, Julia; Steffes, Georg; Erdmann, Ines; Köhrer, Caroline; Sun, Litao; Wagner, Marina; Schäfer, Kerstin; Wang, Guangxia; Koerdt, Sophia N.; Stum, Morgane; RajBhandary, Uttam L.; Thomas, Ulrich; Aberle, Hermann; Burgess, Robert W.; Yang, Xiang-Lei; Dieterich, Daniela; Storkebaum, Erik

    2015-01-01

    Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNAGly aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT. PMID:26138142

  19. Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code

    NASA Astrophysics Data System (ADS)

    Rozov, Alexey; Demeshkina, Natalia; Khusainov, Iskander; Westhof, Eric; Yusupov, Marat; Yusupova, Gulnara

    2016-01-01

    Posttranscriptional modifications at the wobble position of transfer RNAs play a substantial role in deciphering the degenerate genetic code on the ribosome. The number and variety of modifications suggest different mechanisms of action during messenger RNA decoding, of which only a few were described so far. Here, on the basis of several 70S ribosome complex X-ray structures, we demonstrate how Escherichia coli tRNALysUUU with hypermodified 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the wobble position discriminates between cognate codons AAA and AAG, and near-cognate stop codon UAA or isoleucine codon AUA, with which it forms pyrimidine-pyrimidine mismatches. We show that mnm5s2U forms an unusual pair with guanosine at the wobble position that expands general knowledge on the degeneracy of the genetic code and specifies a powerful role of tRNA modifications in translation. Our models consolidate the translational fidelity mechanism proposed previously where the steric complementarity and shape acceptance dominate the decoding mechanism.

  20. Backbone resonance assignments of the m1A22 tRNA methyltransferase TrmK from Bacillus subtilis.

    PubMed

    Dégut, Clément; Barraud, Pierre; Larue, Valéry; Tisné, Carine

    2016-10-01

    RNA modification is a post-transcriptional process by which certain nucleotides are altered after their initial incorporation into an RNA chain. Transfer RNAs (tRNAs) is the most heavily modified class of RNA molecules. These modifications expand the chemical and functional diversity of tRNAs and enhance their structural stability. To date, more than 100 modifications have been identified, the majority of which are specific from one domain of life. However, few modifications are extensively present in the three domains of life. Among those, the m(1)A nucleotide, which consists in the methylation at position 1 of the adenine aromatic ring, is found in tRNAs and ribosomal RNAs. In tRNAs, the m(1)A modification occurs at position 9, 14, 22, 57 and 58. The enzyme TrmK catalyzes the m(1)A formation at position 22. Here we report the backbone (1)H, (15)N and (13)C chemical shift assignments of TrmK from Bacillus subtilis obtained by heteronuclear multidimensional NMR spectroscopy as well as its secondary structure in solution as predicted by TALOS+. These assignments of TrmK pave the way for interaction studies with its tRNA substrates.

  1. Point mutation in mitochondrial tRNA gene is associated with polycystic ovary syndrome and insulin resistance.

    PubMed

    Ding, Yu; Zhuo, Guangchao; Zhang, Caijuan; Leng, Jianhang

    2016-04-01

    Polycystic ovarian syndrome (PCOS) is characterized by chronic anovulation, hyperandrogenism and polycystic ovaries. To date, the molecular mechanisms underlying PCOS have remained to be fully elucidated. As recent studies have revealed a positive association between mitochondrial dysfunction and PCOS, current investigations focus on mutations in the mitochondrial genome of patients with POCS. The present study reported a Chinese patient with PCOS. Sequence analysis of the mitochondrial genome showed the presence of homoplasmic ND5 T12338C and tRNASer (UCN) C7492T mutations as well as a set of polymorphisms belonging to the human mitochondrial haplogroup F2. The T12338C mutation is known to decrease the ND5 mRNA levels and to inhibit the processing of RNA precursors. The C7492T mutation, which occurred at the highly conserved nucleotide in the anticodon stem of the tRNASer (UCN) gene, is important for the tRNA steady‑state level as well as the aminoacylation ability. Therefore, the combination of the ND5 T12338C and tRNASer (UCN) C7492T mutations may lead to mitochondrial dysfunction, and is likely to be involved in the pathogenesis of PCOS. The present study provided novel insight into the molecular mechanisms of PCOS. PMID:26935780

  2. Islander: A database of precisely mapped genomic islands in tRNA and tmRNA genes

    SciTech Connect

    Hudson, Corey M.; Lau, Britney Y.; Williams, Kelly P.

    2014-11-05

    Genomic islands are mobile DNAs that are major agents of bacterial and archaeal evolution. Integration into prokaryotic chromosomes usually occurs site-specifically at tRNA or tmRNA gene (together, tDNA) targets, catalyzed by tyrosine integrases. This splits the target gene, yet sequences within the island restore the disrupted gene; the regenerated target and its displaced fragment precisely mark the endpoints of the island. We applied this principle to search for islands in genomic DNA sequences. Our algorithm identifies tDNAs, finds fragments of those tDNAs in the same replicon and removes unlikely candidate islands through a series of filters. A search for islands in 2168 whole prokaryotic genomes produced 3919 candidates. The website Islander (recently moved to http://bioinformatics.sandia.gov/islander/) presents these precisely mapped candidate islands, the gene content and the island sequence. The algorithm further insists that each island encode an integrase, and attachment site sequence identity is carefully noted; therefore, the database also serves in the study of integrase site-specificity and its evolution.

  3. Islander: A database of precisely mapped genomic islands in tRNA and tmRNA genes

    DOE PAGES

    Hudson, Corey M.; Lau, Britney Y.; Williams, Kelly P.

    2014-11-05

    Genomic islands are mobile DNAs that are major agents of bacterial and archaeal evolution. Integration into prokaryotic chromosomes usually occurs site-specifically at tRNA or tmRNA gene (together, tDNA) targets, catalyzed by tyrosine integrases. This splits the target gene, yet sequences within the island restore the disrupted gene; the regenerated target and its displaced fragment precisely mark the endpoints of the island. We applied this principle to search for islands in genomic DNA sequences. Our algorithm identifies tDNAs, finds fragments of those tDNAs in the same replicon and removes unlikely candidate islands through a series of filters. A search for islandsmore » in 2168 whole prokaryotic genomes produced 3919 candidates. The website Islander (recently moved to http://bioinformatics.sandia.gov/islander/) presents these precisely mapped candidate islands, the gene content and the island sequence. The algorithm further insists that each island encode an integrase, and attachment site sequence identity is carefully noted; therefore, the database also serves in the study of integrase site-specificity and its evolution.« less

  4. ARM-Seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments

    PubMed Central

    Cozen, Aaron E.; Quartley, Erin; Holmes, Andrew D.; Robinson, Eva H.; Phizicky, Eric M.; Lowe, Todd M.

    2015-01-01

    High throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA Methylation sequencing (ARM-Seq) which uses pre-treatment with Escherichia coli AlkB to demethylate 1-methyladenosine, 3-methylcytidine, and 1-methylguanosine, all commonly found in transfer RNAs. Comparative methylation analysis using ARM-Seq provides the first detailed, transcriptome-scale map of these modifications, and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-Seq demonstrates that tRNA-derived small RNAs accurately recapitulate the m1A modification state for well-characterized yeast tRNAs, and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-Seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state, and may reveal new roles for tRNA-derived RNAs as biomarkers or signaling molecules. PMID:26237225

  5. Phylogenetic information from three mitochondrial genomes of Terebelliformia (Annelida) worms and duplication of the methionine tRNA.

    PubMed

    Zhong, Min; Struck, Torsten H; Halanych, Kenneth M

    2008-06-15

    Mitochondrial genomes have been useful for inferring animal phylogeny across a wide range of clades, however they are still poorly sampled in some animal taxa, limiting our knowledge of mtDNA evolution. For example, despite being one of the most diverse animal phyla, only 5 complete annelid mitochrondial genomes have been published. To address this paucity of information, we obtained complete mitochondrial genomic sequences from Pista cristata (Terebellidae) and Terebellides stroemi (Trichobranchidae) as well as one nearly complete mitochondrial genome from Eclysippe vanelli (Ampharetidae). These taxa are within Terebelliformia (Annelida), which include spaghetti worms, icecream cone worms and their relatives. In contrast to the 37 genes found in most bilaterian metazoans, we recover 38 genes in the mitochondrial genomes of T. stroemi and P. cristata due to the presence of a second methionine tRNA (trnM). Interestingly, the two trnMs are located next to each other and are possibly a synapomorphy of these two taxa. The E. vanelli partial mitochondrial genome lacks this additional trnM at the same position, but it may be present in the region not sampled. Compared to other annelids, gene orders of these three mitochondrial genomes are generally conserved except for the atp6-mSSU region. Phylogenetic analyses reveal that mtDNA data strongly supports a Trichobranchidae/Terebellidae clade.

  6. Structure-function relations in the NTPase domain of the antiviral tRNA ribotoxin Escherichia coli PrrC

    SciTech Connect

    Meineke, Birthe; Shuman, Stewart

    2012-06-05

    Breakage of tRNA by Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection. Expression of EcoPrrC is cytocidal in yeast, signifying that PrrC ribotoxicity crosses phylogenetic domain boundaries. EcoPrrC consists of an N-terminal NTPase module that resembles ABC transporters and a C-terminal nuclease module that is sui generis. PrrC homologs are prevalent in many other bacteria. Here we report that Haemophilus influenzae PrrC is toxic in E. coli and yeast. To illuminate structure-activity relations, we conducted a new round of mutational analysis of EcoPrrC guided by primary structure conservation among toxic PrrC homologs. We indentify 17 candidate active site residues in the NTPase module that are essential for toxicity in yeast when EcoPrrC is expressed at high gene dosage. Their functions could be educed by integrating mutational data with the atomic structure of the transition-state complex of a homologous ABC protein.

  7. Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

    PubMed Central

    Fang, Pengfei; Guo, Min

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories. PMID:26670257

  8. Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations.

    PubMed

    Perli, Elena; Fiorillo, Annarita; Giordano, Carla; Pisano, Annalinda; Montanari, Arianna; Grazioli, Paola; Campese, Antonio F; Di Micco, Patrizio; Tuppen, Helen A; Genovese, Ilaria; Poser, Elena; Preziuso, Carmela; Taylor, Robert W; Morea, Veronica; Colotti, Gianni; d'Amati, Giulia

    2016-03-01

    Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNA(Leu(UUR)) or with mutations in the mt-tRNA(Ile), both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs). Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNA(Lys), aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, β30_31 and β32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNA(Leu(UUR)) and mt-tRNA(Lys), and stabilize mutant mt-tRNA(Leu(UUR)). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs.

  9. Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations

    PubMed Central

    Perli, Elena; Fiorillo, Annarita; Giordano, Carla; Pisano, Annalinda; Montanari, Arianna; Grazioli, Paola; Campese, Antonio F.; Di Micco, Patrizio; Tuppen, Helen A.; Genovese, Ilaria; Poser, Elena; Preziuso, Carmela; Taylor, Robert W.; Morea, Veronica; Colotti, Gianni; d'Amati, Giulia

    2016-01-01

    Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNALeu(UUR) or with mutations in the mt-tRNAIle, both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs). Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNALys, aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, β30_31 and β32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNALeu(UUR) and mt-tRNALys, and stabilize mutant mt-tRNALeu(UUR). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs. PMID:26721932

  10. Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

    PubMed

    Bar-Yaacov, Dan; Frumkin, Idan; Yashiro, Yuka; Chujo, Takeshi; Ishigami, Yuma; Chemla, Yonatan; Blumberg, Amit; Schlesinger, Orr; Bieri, Philipp; Greber, Basil; Ban, Nenad; Zarivach, Raz; Alfonta, Lital; Pilpel, Yitzhak; Suzuki, Tsutomu; Mishmar, Dan

    2016-09-01

    The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function. PMID:27631568

  11. Utp22p acts in concert with Utp8p to channel aminoacyl-tRNA from the nucleolus to the nuclear tRNA export receptor Los1p but not Msn5p.

    PubMed

    Eswara, Manoja B K; Clayton, Ashley; Mangroo, Dev

    2012-12-01

    Utp8p is an essential nucleolar protein that channels aminoacyl-tRNAs from aminoacyl-tRNA synthetases in the nucleolus to the nuclear tRNA export receptors located in the nucleoplasm and nuclear pore complex in Saccharomyces cerevisiae. Utp8p is also part of the U3 snoRNA-associated protein complex involved in 18S rRNA biogenesis in the nucleolus. We report that Utp22p, which is another member of the U3 snoRNA-associated protein complex, is also an intranuclear component of the nuclear tRNA export machinery. Depletion of Utp22p results in nuclear retention of mature tRNAs derived from intron-containing and intronless precursors. Moreover, Utp22p copurifies with the nuclear tRNA export receptor Los1p, the aminoacyl-tRNA synthetase Tys1p and Utp8p, but not with the RanGTPase Gsp1p and the nuclear tRNA export receptor Msn5p. Utp22p interacts directly with Utp8p and Los1p in a tRNA-independent manner in vitro. Utp22p also interacts directly with Tys1p, but this binding is stimulated when Tys1p is bound to tRNA. However, Utp22p, unlike Utp8p, does not bind tRNA saturably. These data suggest that Utp22p recruits Utp8p to aminoacyl-tRNA synthetases in the nucleolus to collect aminoacyl-tRNA and then accompanies the Utp8p-tRNA complex to deliver the aminoacyl-tRNAs to Los1p but not Msn5p. It is possible that Nrap/Nol6, the mammalian orthologue of Utp22p, plays a role in channelling aminoacyl-tRNA to the nuclear tRNA export receptor exportin-t.

  12. The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

    PubMed Central

    Padilla-Mejía, Norma E.; Florencio-Martínez, Luis E.; Moreno-Campos, Rodrigo; Vizuet-de-Rueda, Juan C.; Cevallos, Ana M.; Hernández-Rivas, Rosaura; Manning-Cela, Rebeca

    2014-01-01

    Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major. This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5′ end of all the mRNAs. In L. major, tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5′ rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5′ end and a poly(A) tail at the 3′ end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei. However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo, as has recently been found in other eukaryotes. PMID:25548151

  13. Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system

    PubMed Central

    Wan, Leo C. K.; Mao, Daniel Y. L.; Neculai, Dante; Strecker, Jonathan; Chiovitti, David; Kurinov, Igor; Poda, Gennadiy; Thevakumaran, Neroshan; Yuan, Fang; Szilard, Rachel K.; Lissina, Elena; Nislow, Corey; Caudy, Amy A.; Durocher, Daniel; Sicheri, Frank

    2013-01-01

    The universally conserved Kae1/Qri7/YgjD and Sua5/YrdC protein families have been implicated in growth, telomere homeostasis, transcription and the N6-threonylcarbamoylation (t6A) of tRNA, an essential modification required for translational fidelity by the ribosome. In bacteria, YgjD orthologues operate in concert with the bacterial-specific proteins YeaZ and YjeE, whereas in archaeal and eukaryotic systems, Kae1 operates as part of a larger macromolecular assembly called KEOPS with Bud32, Cgi121, Gon7 and Pcc1 subunits. Qri7 orthologues function in the mitochondria and may represent the most primitive member of the Kae1/Qri7/YgjD protein family. In accordance with previous findings, we confirm that Qri7 complements Kae1 function and uncover that Qri7 complements the function of all KEOPS subunits in growth, t6A biosynthesis and, to a partial degree, telomere maintenance. These observations suggest that Kae1 provides a core essential function that other subunits within KEOPS have evolved to support. Consistent with this inference, Qri7 alone is sufficient for t6A biosynthesis with Sua5 in vitro. In addition, the 2.9 Å crystal structure of Qri7 reveals a simple homodimer arrangement that is supplanted by the heterodimerization of YgjD with YeaZ in bacteria and heterodimerization of Kae1 with Pcc1 in KEOPS. The partial complementation of telomere maintenance by Qri7 hints that KEOPS has evolved novel functions in higher organisms. PMID:23620299

  14. Reconstitution and characterization of eukaryotic N6-threonylcarbamoylation of tRNA using a minimal enzyme system.

    PubMed

    Wan, Leo C K; Mao, Daniel Y L; Neculai, Dante; Strecker, Jonathan; Chiovitti, David; Kurinov, Igor; Poda, Gennadiy; Thevakumaran, Neroshan; Yuan, Fang; Szilard, Rachel K; Lissina, Elena; Nislow, Corey; Caudy, Amy A; Durocher, Daniel; Sicheri, Frank

    2013-07-01

    The universally conserved Kae1/Qri7/YgjD and Sua5/YrdC protein families have been implicated in growth, telomere homeostasis, transcription and the N6-threonylcarbamoylation (t(6)A) of tRNA, an essential modification required for translational fidelity by the ribosome. In bacteria, YgjD orthologues operate in concert with the bacterial-specific proteins YeaZ and YjeE, whereas in archaeal and eukaryotic systems, Kae1 operates as part of a larger macromolecular assembly called KEOPS with Bud32, Cgi121, Gon7 and Pcc1 subunits. Qri7 orthologues function in the mitochondria and may represent the most primitive member of the Kae1/Qri7/YgjD protein family. In accordance with previous findings, we confirm that Qri7 complements Kae1 function and uncover that Qri7 complements the function of all KEOPS subunits in growth, t(6)A biosynthesis and, to a partial degree, telomere maintenance. These observations suggest that Kae1 provides a core essential function that other subunits within KEOPS have evolved to support. Consistent with this inference, Qri7 alone is sufficient for t(6)A biosynthesis with Sua5 in vitro. In addition, the 2.9 Å crystal structure of Qri7 reveals a simple homodimer arrangement that is supplanted by the heterodimerization of YgjD with YeaZ in bacteria and heterodimerization of Kae1 with Pcc1 in KEOPS. The partial complementation of telomere maintenance by Qri7 hints that KEOPS has evolved novel functions in higher organisms.

  15. Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

    SciTech Connect

    Ling, Jiqiang; Peterson, Kaitlyn M.; Simonovic, Ivana; Cho, Chris; Soll, Dieter; Simonovic, Miljan

    2014-03-12

    Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular ?? with a threonine (Thr) anticodon, MST1 also recognizes an unusual ??, which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employs distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for ?? recognition, whereas the anticodon sequence is essential for aminoacylation of ??. The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate ??, reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix {alpha}11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.

  16. Hyaloraphidium curvatum: a linear mitochondrial genome, tRNA editing, and an evolutionary link to lower fungi.

    PubMed

    Forget, Lise; Ustinova, Jana; Wang, Zhang; Huss, Volker A R; Lang, B Franz

    2002-03-01

    We have sequenced the mitochondrial DNA (mtDNA) of Hyaloraphidium curvatum, an organism previously classified as a colorless green alga but now recognized as a lower fungus based on molecular data. The 29.97-kbp mitochondrial chromosome is maintained as a monomeric, linear molecule with identical, inverted repeats (1.43 kbp) at both ends, a rare genome architecture in mitochondria. The genome encodes only 14 known mitochondrial proteins, 7 tRNAs, the large subunit rRNA and small subunit rRNA (SSU rRNA), and 3 ORFs. The SSU rRNA is encoded in two gene pieces that are located 8 kbp apart on the mtDNA. Scrambled and fragmented mitochondrial rRNAs are well known from green algae and alveolate protists but are unprecedented in fungi. Protein genes code for apocytochrome b; cytochrome oxidase 1, 2, and 3, NADH dehydrogenase 1, 2, 3, 4, 4L, 5, and 6, and ATP synthase 6, 8, and 9 subunits, and several of these genes are organized in operon-like clusters. The set of seven mitochondrially encoded tRNAs is insufficient to recognize all codons that occur in the mitochondrial protein genes. When taking into account the pronounced codon bias, at least 16 nuclear-encoded tRNAs are assumed to be imported into the mitochondria. Three of the seven predicted mitochondria-encoded tRNA sequences carry mispairings in the first three positions of the acceptor stem. This strongly suggests that these tRNAs are edited by a mechanism similar to the one seen in the fungus Spizellomyces punctatus and the rhizopod amoeba Acanthamoeba castellanii. Our phylogenetic analysis confirms with overwhelming support that H. curvatum is a member of the chytridiomycete fungi, specifically related to the Monoblepharidales.

  17. G-quadruplex structures contribute to the neuroprotective effects of angiogenin-induced tRNA fragments

    PubMed Central

    Ivanov, Pavel; O’Day, Elizabeth; Emara, Mohamed M.; Wagner, Gerhard; Lieberman, Judy; Anderson, Paul

    2014-01-01

    Angiogenin (ANG) is a stress-activated ribonuclease that promotes the survival of motor neurons. Ribonuclease inactivating point mutations are found in a subset of patients with ALS, a fatal neurodegenerative disease with no cure. We recently showed that ANG cleaves tRNA within anticodon loops to produce 5′- and 3′-fragments known as tRNA-derived, stress-induced RNAs (tiRNAs). Selected 5′-tiRNAs (e.g., tiRNAAla, tiRNACys) cooperate with the translational repressor Y-box binding protein 1 (YB-1) to displace the cap-binding complex eIF4F from capped mRNA, inhibit translation initiation, and induce the assembly of stress granules (SGs). Here, we show that translationally active tiRNAs assemble unique G-quadruplex (G4) structures that are required for translation inhibition. We show that tiRNAAla binds the cold shock domain of YB-1 to activate these translational reprogramming events. We discovered that 5′-tiDNAAla (the DNA equivalent of 5′-tiRNAAla) is a stable tiRNA analog that displaces eIF4F from capped mRNA, inhibits translation initiation, and induces the assembly of SGs. The 5′-tiDNAAla also assembles a G4 structure that allows it to enter motor neurons spontaneously and trigger a neuroprotective response in a YB-1–dependent manner. Remarkably, the ability of 5′-tiRNAAla to induce SG assembly is inhibited by G4 structures formed by pathological GGGGCC repeats found in C9ORF72, the most common genetic cause of ALS, suggesting that functional interactions between G4 RNAs may contribute to neurodegenerative disease. PMID:25404306

  18. Post-transcriptional Boolean computation by combining aptazymes controlling mRNA translation initiation and tRNA activation.

    PubMed

    Klauser, Benedikt; Saragliadis, Athanasios; Ausländer, Simon; Wieland, Markus; Berthold, Michael R; Hartig, Jörg S

    2012-09-01

    In cellular systems environmental and metabolic signals are integrated for the conditional control of gene expression. On the other hand, artificial manipulation of gene expression is of high interest for metabolic and genetic engineering. Especially the reprogramming of gene expression patterns to orchestrate cellular responses in a predictable fashion is considered to be of great importance. Here we introduce a highly modular RNA-based system for performing Boolean logic computation at a post-transcriptional level in Escherichia coli. We have previously shown that artificial riboswitches can be constructed by utilizing ligand-dependent Hammerhead ribozymes (aptazymes). Employing RNA self-cleavage as the expression platform-mechanism of an artificial riboswitch has the advantage that it can be applied to control several classes of RNAs such as mRNAs, tRNAs, and rRNAs. Due to the highly modular and orthogonal nature of these switches it is possible to combine aptazyme regulation of activating a suppressor tRNA with the regulation of mRNA translation initiation. The different RNA classes can be controlled individually by using distinct aptamers for individual RNA switches. Boolean logic devices are assembled by combining such switches in order to act on the expression of a single mRNA. In order to demonstrate the high modularity, a series of two-input Boolean logic operators were constructed. For this purpose, we expanded our aptazyme toolbox with switches comprising novel behaviours with respect to the small molecule triggers thiamine pyrophosphate (TPP) and theophylline. Then, individual switches were combined to yield AND, NOR, and ANDNOT gates. This study demonstrates that post-transcriptional aptazyme-based switches represent versatile tools for engineering advanced genetic devices and circuits without the need for regulatory protein cofactors. PMID:22777205

  19. Structure-guided discovery of carboxy-SAM as a novel metabolite modulating tRNA function

    PubMed Central

    Kim, Jungwook; Xiao, Hui; Bonanno, Jeffrey B.; Kalyanaraman, Chakrapani; Brown, Shoshana; Tang, Xiangying; Al-Obaidi, Nawar F.; Patskovsky, Yury; Babbitt, Patricia C.; Jacobson, Matthew P.; Lee, Young-Sam; Almo, Steven C.

    2014-01-01

    Identifying novel metabolites and characterizing their biological functions are major challenges of the post-genomic era. X-ray crystallography can reveal unanticipated ligands which persist through purification and crystallization. These adventitious protein:ligand complexes provide insights into new activities, pathways and regulatory mechanisms. We describe a new metabolite, carboxy-S-adenosylmethionine (Cx-SAM), its biosynthetic pathway and its role in tRNA modification. The structure of CmoA, a member of the SAM-dependent methyltransferase superfamily, revealed a ligand in the catalytic site consistent with Cx-SAM. Mechanistic analyses demonstrated an unprecedented role for prephenate as the carboxyl donor and the involvement of a unique ylide intermediate as the carboxyl acceptor in the CmoA-mediated conversion of SAM to Cx-SAM. A second member of the SAM-dependent methyltransferase superfamily, CmoB, recognizes Cx-SAM and acts as a carboxymethyltransferase to convert 5-hydroxyuridine (ho5U) into 5-oxyacetyl uridine (cmo5U) at the wobble position of multiple tRNAs in Gram negative bacteria1, resulting in expanded codon-recognition properties2,3. CmoA and CmoB represent the first documented synthase and transferase for Cx-SAM. These findings reveal new functional diversity in the SAM-dependent methyltransferase superfamily and expand the metabolic and biological contributions of SAM-based biochemistry. These discoveries highlight the value of structural genomics approaches for identifying ligands in the context of their physiologically relevant macromolecular binding partners and for aiding in functional assignment. PMID:23676670

  20. The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK).

    PubMed

    Roovers, Martine; Kaminska, Katarzyna H; Tkaczuk, Karolina L; Gigot, Daniel; Droogmans, Louis; Bujnicki, Janusz M

    2008-06-01

    N(1)-methylation of adenosine to m(1)A occurs in several different positions in tRNAs from various organisms. A methyl group at position N(1) prevents Watson-Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules. Thus far, only one family of genes encoding enzymes responsible for m(1)A methylation at position 58 has been identified, while other m(1)A methyltransferases (MTases) remain elusive. Here, we show that Bacillus subtilis open reading frame yqfN is necessary and sufficient for N(1)-adenosine methylation at position 22 of bacterial tRNA. Thus, we propose to rename YqfN as TrmK, according to the traditional nomenclature for bacterial tRNA MTases, or TrMet(m(1)A22) according to the nomenclature from the MODOMICS database of RNA modification enzymes. tRNAs purified from a DeltatrmK strain are a good substrate in vitro for the recombinant TrmK protein, which is sufficient for m(1)A methylation at position 22 as are tRNAs from Escherichia coli, which natively lacks m(1)A22. TrmK is conserved in Gram-positive bacteria and present in some Gram-negative bacteria, but its orthologs are apparently absent from archaea and eukaryota. Protein structure prediction indicates that the active site of TrmK does not resemble the active site of the m(1)A58 MTase TrmI, suggesting that these two enzymatic activities evolved independently.

  1. Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus

    PubMed Central

    Ishida, Kazuo; Kunibayashi, Takashi; Tomikawa, Chie; Ochi, Anna; Kanai, Tamotsu; Hirata, Akira; Iwashita, Chikako; Hori, Hiroyuki

    2011-01-01

    Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50°C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50°C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m5s2U54 and m1A58 in tRNA from the ΔtruB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the Ψ55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the Ψ55 modification is required for low-temperature adaptation to control other modified. 35S-Met incorporation analysis showed that the protein synthesis activity of the ΔtruB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ΔtruB cells at 50°C. PMID:21097467

  2. Defects in tRNA Anticodon Loop 2'-O-Methylation Are Implicated in Nonsyndromic X-Linked Intellectual Disability due to Mutations in FTSJ1.

    PubMed

    Guy, Michael P; Shaw, Marie; Weiner, Catherine L; Hobson, Lynne; Stark, Zornitza; Rose, Katherine; Kalscheuer, Vera M; Gecz, Jozef; Phizicky, Eric M

    2015-12-01

    tRNA modifications are crucial for efficient and accurate protein synthesis, and modification defects are frequently associated with disease. Yeast trm7Δ mutants grow poorly due to lack of 2'-O-methylated C32 (Cm32 ) and Gm34 on tRNA(Phe) , catalyzed by Trm7-Trm732 and Trm7-Trm734, respectively, which in turn results in loss of wybutosine at G37 . Mutations in human FTSJ1, the likely TRM7 homolog, cause nonsyndromic X-linked intellectual disability (NSXLID), but the role of FTSJ1 in tRNA modification is unknown. Here, we report that tRNA(Phe) from two genetically independent cell lines of NSXLID patients with loss-of-function FTSJ1 mutations nearly completely lacks Cm32 and Gm34 , and has reduced peroxywybutosine (o2yW37 ). Additionally, tRNA(Phe) from an NSXLID patient with a novel FTSJ1-p.A26P missense allele specifically lacks Gm34 , but has normal levels of Cm32 and o2yW37 . tRNA(Phe) from the corresponding Saccharomyces cerevisiae trm7-A26P mutant also specifically lacks Gm34 , and the reduced Gm34 is not due to weaker Trm734 binding. These results directly link defective 2'-O-methylation of the tRNA anticodon loop to FTSJ1 mutations, suggest that the modification defects cause NSXLID, and may implicate Gm34 of tRNA(Phe) as the critical modification. These results also underscore the widespread conservation of the circuitry for Trm7-dependent anticodon loop modification of eukaryotic tRNA(Phe) . PMID:26310293

  3. Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus.

    PubMed

    Ishida, Kazuo; Kunibayashi, Takashi; Tomikawa, Chie; Ochi, Anna; Kanai, Tamotsu; Hirata, Akira; Iwashita, Chikako; Hori, Hiroyuki

    2011-03-01

    Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50 °C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50 °C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m(5)s(2)U54 and m(1)A58 in tRNA from the ΔtruB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the Ψ55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the Ψ55 modification is required for low-temperature adaptation to control other modified. (35)S-Met incorporation analysis showed that the protein synthesis activity of the ΔtruB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ΔtruB cells at 50°C.

  4. tRNomics: analysis of tRNA genes from 50 genomes of Eukarya, Archaea, and Bacteria reveals anticodon-sparing strategies and domain-specific features.

    PubMed Central

    Marck, Christian; Grosjean, Henri

    2002-01-01

    From 50 genomes of the three domains of life (7 eukarya, 13 archaea, and 30 bacteria), we extracted, analyzed, and compared over 4,000 sequences corresponding to cytoplasmic, nonorganellar tRNAs. For each genome, the complete set of tRNAs required to read the 61 sense codons was identified, which permitted revelation of three major anticodon-sparing strategies. Other features and sequence peculiarities analyzed are the following: (1) fit to the standard cloverleaf structure, (2) characteristic consensus sequences for elongator and initiator tDNAs, (3) frequencies of bases at each sequence position, (4) type and frequencies of conserved 2D and 3D base pairs, (5) anticodon/tDNA usages and anticodon-sparing strategies, (6) identification of the tRNA-Ile with anticodon CAU reading AUA, (7) size of variable arm, (8) occurrence and location of introns, (9) occurrence of 3'-CCA and 5'-extra G encoded at the tDNA level, and (10) distribution of the tRNA genes in genomes and their mode of transcription. Among all tRNA isoacceptors, we found that initiator tDNA-iMet is the most conserved across the three domains, yet domain-specific signatures exist. Also, according to which tRNA feature is considered (5'-extra G encoded in tDNAs-His, AUA codon read by tRNA-Ile with anticodon CAU, presence of intron, absence of "two-out-of-three" reading mode and short V-arm in tDNA-Tyr) Archaea sequester either with Bacteria or Eukarya. No common features between Eukarya and Bacteria not shared with Archaea could be unveiled. Thus, from the tRNomic point of view, Archaea appears as an "intermediate domain" between Eukarya and Bacteria. PMID:12403461

  5. Structural modeling identified the tRNA-binding domain of Utp8p, an essential nucleolar component of the nuclear tRNA export machinery of Saccharomyces cerevisiae.

    PubMed

    McGuire, Andrew T; Keates, Robert A B; Cook, Stephanie; Mangroo, Dev

    2009-04-01

    Utp8p is an essential 80 kDa intranuclear tRNA chaperone that transports tRNAs from the nucleolus to the nuclear tRNA export receptors in Saccharomyces cerevisiae. To help understand the mechanism of Utp8p function, predictive tools were used to derive a partial model of the tertiary structure of Utp8p. Secondary structure prediction, supported by circular dichroism measurements, indicated that Utp8p is divided into 2 domains: the N-terminal beta sheet and the C-terminal alpha helical domain. Tertiary structure prediction was more challenging, because the amino acid sequence of Utp8p is not directly homologous to any known protein structure. The tertiary structures predicted by threading and fold recognition had generally modest scores, but for the C-terminal domain, threading and fold recognition consistently pointed to an alpha-alpha superhelix. Because of the sequence diversity of this fold type, no single structural template was an ideal fit to the Utp8p sequence. Instead, a composite template was constructed from 3 different alpha-alpha superhelix structures that gave the best matches to different portions of the C-terminal domain sequence. In the resulting model, the most conserved sequences grouped in a tight cluster of positive charges on a protein that is otherwise predominantly negative, suggesting that the positive-charge cleft may be the tRNA-binding site. Mutations of conserved positive residues in the proposed binding site resulted in a reduction in the affinity of Utp8p for tRNA both in vivo and in vitro. Models were also derived for the 10 fungal homologues of Utp8p, and the localization of the positive charges on the conserved surface was found in all cases. Taken together, these data suggest that the positive-charge cleft of the C-terminal domain of Utp8p is involved in tRNA-binding.

  6. A novel rearrangement in the mitochondrial genome of tongue sole, Cynoglossus semilaevis: control region translocation and a tRNA gene inversion.

    PubMed

    Kong, Xiaoyu; Dong, Xiaoli; Zhang, Yanchun; Shi, Wei; Wang, Zhongming; Yu, Ziniu

    2009-12-01

    The organization of fish mitochondrial genomes (mitogenomes) is quite conserved, usually with the heavy strand encoding 12 of 13 protein-coding genes and 14 of 22 tRNA genes, and the light strand encoding ND6 and the remaining 8 tRNA genes. Currently, there are only a few reports on gene reorganization of fish mitogenomes, with only two types of rearrangements (shuffling and translocation) observed. No gene inversion has been detected in approximately 420 complete fish mitogenomes available so far. Here we report a novel rearrangement in the mitogenome of Cynoglossus semilaevis (Cynoglossinae, Cynoglossidae, Pleuronectiformes). The genome is 16,371 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 2 main noncoding regions, the putative control region and the light-strand replication origin. A striking finding of this study is that the tRNA(Gln) gene is translocated from the light to the heavy strand (Q inversion). This is accompanied by shuffling of the tRNA(Ile) gene and long-range translocation of the putative control region downstream to a site between ND1 and the tRNA(Gln) gene. The remaining gene order is identical to that of typical fish mitogenomes. Additionally, unique characters of this mitogenome, including a high A+T content and length variations of 8 protein-coding genes, were found through comparison of the mitogenome sequence with those from other flatfishes. All the features detected and their relationships with the rearrangements, as well as a possible rearrangement pathway, are discussed. These data provide interesting information for better understanding the molecular mechanisms of gene reorganization in fish mitogenomes.

  7. Allele-specific suppressors of lin-1(R175Opal) identify functions of MOC-3 and DPH-3 in tRNA modification complexes in Caenorhabditis elegans.

    PubMed

    Kim, Sunhong; Johnson, Wade; Chen, Changchun; Sewell, Aileen K; Byström, Anders S; Han, Min

    2010-08-01

    The elongator (ELP) complex consisting of Elp1-6p has been indicated to play roles in multiple cellular processes. In yeast, the ELP complex has been shown to genetically interact with Uba4p/Urm1p and Kti11-13p for a function in tRNA modification. Through a Caenorhabditis elegans genetic suppressor screen and positional cloning, we discovered that loss-of-function mutations of moc-3 and dph-3, orthologs of the yeast UBA4 and KTI11, respectively, effectively suppress the Multivulva (Muv) phenotype of the lin-1(e1275, R175Opal) mutation. These mutations do not suppress the Muv phenotype caused by other lin-1 alleles or by gain-of-function alleles of ras or raf that act upstream of lin-1. The suppression can also be reverted by RNA interference of lin-1. Furthermore, we showed that dph-3(lf) also suppressed the defect of lin-1(e1275) in promoting the expression of a downstream target (egl-17). These results indicate that suppression by the moc-3 and dph-3 mutations is due to the elevated activity of lin-1(e1275) itself rather than the altered activity of a factor downstream of lin-1. We further showed that loss-of-function mutations of urm-1 and elpc-1-4, the worm counterparts of URM1 and ELP complex components in yeast, also suppressed lin-1(e1275). We also confirmed that moc-3(lf) and dph-3(lf) have defects in tRNA modifications as do the mutants of their yeast orthologs. These results, together with the observation of a likely readthrough product from a lin-1(e1275)::gfp fusion transgene indicate that the aberrant tRNA modification led to failed recognition of a premature stop codon in lin-1(e1275). Our genetic data suggest that the functional interaction of moc-3/urm-1 and dph-3 with the ELP complex is an evolutionarily conserved mechanism involved in tRNA functions that are important for accurate translation. PMID:20479142

  8. Impact of P-Site tRNA and Antibiotics on Ribosome Mediated Protein Folding: Studies Using the Escherichia coli Ribosome

    PubMed Central

    Mondal, Surojit; Pathak, Bani Kumar; Ray, Sutapa; Barat, Chandana

    2014-01-01

    Background The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity. Results The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′–CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability. Conclusion Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions. PMID:25000563

  9. X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold.

    PubMed

    Ericsson, Ulrika B; Nordlund, Pär; Hallberg, B Martin

    2004-05-01

    Pseudouridine synthases catalyse the isomerisation of uridine to pseudouridine in structural RNA. The pseudouridine synthase TruD, that modifies U13 in tRNA, belongs to a recently identified and large family of pseudouridine synthases present in all kingdoms of life. We report here the crystal structure of Escherichia coli TruD at 2.0 A resolution. The structure reveals an overall V-shaped molecule with an RNA-binding cleft formed between two domains: a catalytic domain and an insertion domain. The catalytic domain has a fold similar to that of the catalytic domains of previously characterised pseudouridine synthases, whereas the insertion domain displays a novel fold.

  10. [Analysis of nucleotide sequences polymorphism of chloroplast trnL-trnF spacer of tRNA genes in giant duckweed Spirodela polyrrhiza (L.) Schleiden].

    PubMed

    Ryzhova, N N; Martirosian, L V; Kolganova, T V; Goriunova, S V; Kochieva, E Z

    2006-01-01

    Chloroplast DNA trnL-trnF spacer sequences of tRNA genes of 14 specimens of the fam. Lemnaceae have been characterized. Nucleotide polymorphism analysis of the spacer trnL-trnF of geographically isolated and morphologically differing accessions of S. polyrrhiza that is the most widespread species of Spirodela genus showed the low level of intraspecific variability. Five trnL-trnF haplotypes of S. polyrrhiza are identified. Both mono-, and polynucleotide repeats, and also extensive indels, specific to representatives Spirodela polyrrhiza, Landoltia punctata and Lemna sp. are revealed. Competency of Landoltia genus allocation as separate entity was confirmed. PMID:17209426

  11. One ancestor for two codes viewed from the perspective of two complementary modes of tRNA aminoacylation

    PubMed Central

    Rodin, Andrei S; Szathmáry, Eörs; Rodin, Sergei N

    2009-01-01

    Background The genetic code is brought into action by 20 aminoacyl-tRNA synthetases. These enzymes are evenly divided into two classes (I and II) that recognize tRNAs from the minor and major groove sides of the acceptor stem, respectively. We have reported recently that: (1) ribozymic precursors of the synthetases seem to have used the same two sterically mirror modes of tRNA recognition, (2) having these two modes might have helped in preventing erroneous aminoacylation of ancestral tRNAs with complementary anticodons, yet (3) the risk of confusion for the presumably earliest pairs of complementarily encoded amino acids had little to do with anticodons. Accordingly, in this communication we focus on the acceptor stem. Results Our main result is the emergence of a palindrome structure for the acceptor stem's common ancestor, reconstructed from the phylogenetic trees of Bacteria, Archaea and Eukarya. In parallel, for pairs of ancestral tRNAs with complementary anticodons, we present updated evidence of concerted complementarity of the second bases in the acceptor stems. These two results suggest that the first pairs of "complementary" amino acids that were engaged in primordial coding, such as Gly and Ala, could have avoided erroneous aminoacylation if and only if the acceptor stems of their adaptors were recognized from the same, major groove, side. The class II protein synthetases then inherited this "primary preference" from isofunctional ribozymes. Conclusion Taken together, our results support the hypothesis that the genetic code per se (the one associated with the anticodons) and the operational code of aminoacylation (associated with the acceptor) diverged from a common ancestor that probably began developing before translation. The primordial advantage of linking some amino acids (most likely glycine and alanine) to the ancestral acceptor stem may have been selective retention in a protocell surrounded by a leaky membrane for use in nucleotide and coenzyme

  12. TAP1, a yeast gene that activates the expression of a tRNA gene with a defective internal promoter.

    PubMed Central

    Di Segni, G; McConaughy, B L; Shapiro, R A; Aldrich, T L; Hall, B D

    1993-01-01

    We developed a genetic selection system based on nonsense suppression in Saccharomyces cerevisiae to identify mutations in proteins involved in transcription initiation by RNA polymerase III. A SUP4 tRNA(Tyr) internal promoter mutation (A53T61) that was unable to suppress ochre mutations in vivo and was incapable of binding TFIIIC in vitro was used as the target for selection of trans-acting compensatory mutations. We identified two such mutations in the same gene, which we named TAP1 (for transcription activation protein). The level of the SUP4A53T61 transcript was threefold higher in the tap1-1 mutant than in the wild type. The tap1-1 mutant strain was also temperature sensitive for growth. The thermosensitive character cosegregated with the restorer of suppression activity, as shown by meiotic linkage analysis and coreversion of the two traits. At 1 to 2 h after a shift to the restrictive temperature, RNA synthesis was strongly inhibited in the tap1-1 mutant, preceding any effect upon protein synthesis or growth. A marked decrease in tRNA and 5S rRNA synthesis was seen, and shortly after that, rRNA synthesis was inhibited. By complementation of the ts- growth defect, we cloned the wild-type TAP1 gene. It is essential for yeast growth. We show in the accompanying report (T. L. Aldrich, G. Di Segni, B. L. McConaughy, N. J. Keen, S. Whelen, and B. D. Hall, Mol. Cell. Biol. 13:3434-3444, 1993) that TAP1 is identical to RAT1, a yeast gene implicated in poly(A)+ RNA export and that the TAP1/RAT1 gene product has extensive sequence similarity to the protein encoded by another yeast gene (variously named DST2, KEM1, RAR5, SEP1, or XRN1) having exonuclease and DNA strand transfer activity (reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]). Images PMID:8497259

  13. Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotypes

    PubMed Central

    Pope, Welkin H.; Anders, Kirk R.; Baird, Madison; Bowman, Charles A.; Boyle, Michelle M.; Broussard, Gregory W.; Chow, Tiffany; Clase, Kari L.; Cooper, Shannon; Cornely, Kathleen A.; DeJong, Randall J.; Delesalle, Veronique A.; Deng, Lisa; Dunbar, David; Edgington, Nicholas P.; Ferreira, Christina M.; Weston Hafer, Kathleen; Hartzog, Grant A.; Hatherill, J. Robert; Hughes, Lee E.; Ipapo, Khristina; Krukonis, Greg P.; Meier, Christopher G.; Monti, Denise L.; Olm, Matthew R.; Page, Shallee T.; Peebles, Craig L.; Rinehart, Claire A.; Rubin, Michael R.; Russell, Daniel A.; Sanders, Erin R.; Schoer, Morgan; Shaffer, Christopher D.; Wherley, James; Vazquez, Edwin; Yuan, Han; Zhang, Daiyuan; Cresawn, Steven G.; Jacobs-Sera, Deborah; Hendrix, Roger W.

    2014-01-01

    ABSTRACT Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc2155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages

  14. Evolution of Pulmonate Gastropod Mitochondrial Genomes: Comparisons of Gene Organizations of Euhadra, Cepaea and Albinaria and Implications of Unusual Trna Secondary Structures

    PubMed Central

    Yamazaki, N.; Ueshima, R.; Terrett, J. A.; Yokobori, S. I.; Kaifu, M.; Segawa, R.; Kobayashi, T.; Numachi, K. I.; Ueda, T.; Nishikawa, K.; Watanabe, K.; Thomas, R. H.

    1997-01-01

    Complete gene organizations of the mitochondrial genomes of three pulmonate gastropods, Euhadra herklotsi, Cepaea nemoralis and Albinaria coerulea, permit comparisons of their gene organizations. Euhadra and Cepaea are classified in the same superfamily, Helicoidea, yet they show several differences in the order of tRNA and protein coding genes. Albinaria is distantly related to the other two genera but shares the same gene order in one part of its mitochondrial genome with Euhadra and in another part with Cepaea. Despite their small size (14.1-14.5 kbp), these snail mtDNAs encode 13 protein genes, two rRNA genes and at least 22 tRNA genes. These genomes exhibit several unusual or unique features compared to other published metazoan mitochondrial genomes, including those of other molluscs. Several tRNAs predicted from the DNA sequences possess bizarre structures lacking either the T stem or the D stem, similar to the situation seen in nematode mt-tRNAs. The acceptor stems of many tRNAs show a considerable number of mismatched basepairs, indicating that the RNA editing process recently demonstrated in Euhadra is widespread in the pulmonate gastropods. Strong selection acting on mitochondrial genomes of these animals would have resulted in frequent occurrence of the mismatched basepairs in regions of overlapping genes. PMID:9055084

  15. Yeast H2A.Z, FACT complex and RSC regulate transcription of tRNA gene through differential dynamics of flanking nucleosomes.

    PubMed

    Mahapatra, Sahasransu; Dewari, Pooran S; Bhardwaj, Anubhav; Bhargava, Purnima

    2011-05-01

    FACT complex is involved in elongation and ensures fidelity in the initiation step of transcription by RNA polymerase (pol) II. Histone variant H2A.Z is found in nucleosomes at the 5'-end of many genes. We report here H2A.Z-chaperone activity of the yeast FACT complex on the short, nucleosome-free, non-coding, pol III-transcribed yeast tRNA genes. On a prototype gene, yeast SUP4, chromatin remodeler RSC and FACT regulate its transcription through novel mechanisms, wherein the two gene-flanking nucleosomes containing H2A.Z, play different roles. Nhp6, which ensures transcription fidelity and helps load yFACT onto the gene flanking nucleosomes, has inhibitory role. RSC maintains a nucleosome abutting the gene terminator downstream, which results in reduced transcription rate in active state while H2A.Z probably helps RSC in keeping the gene nucleosome-free and serves as stress-sensor. All these factors maintain an epigenetic state which allows the gene to return quickly from repressed to active state and tones down the expression from the active SUP4 gene, required probably to maintain the balance in cellular tRNA pool.

  16. Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis.

    PubMed

    Purta, Elzbieta; van Vliet, Françoise; Tricot, Catherine; De Bie, Lara G; Feder, Marcin; Skowronek, Krzysztof; Droogmans, Louis; Bujnicki, Janusz M

    2005-05-15

    The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.

  17. Congenital encephalomyopathy and adult-onset myopathy and diabetes mellitus: Different phenotypic associations of a new heteroplasmic mtDNA tRNA glutamic acid mutation

    SciTech Connect

    Hanna, M.G.; Nelson, I.; Sweeney, M.G.; Cooper, J.M.; Watkins, P.J.; Morgan-Hughes, J.A.; Harding, A.E.

    1995-05-01

    We report the clinical, biochemical, and molecular genetic findings in a family with an unusual mitochondrial disease phenotype harboring a novel mtDNA tRNA glutamic acid mutation at position 14709. The proband and his sister presented with congenital myopathy and mental retardation and subsequently developed cerebellar ataxia. Other family members had either adult-onset diabetes mellitus with muscle weakness or adult-onset diabetes mellitus alone. Ragged-red and cytochrome c oxidase (COX)-negative fibers were present in muscle biopsies. Biochemical studies of muscle mitochondria showed reduced complex I and IV activities. The mtDNA mutation was heteroplasmic in blood and muscle in all matrilineal relatives analyzed. Primary myoblast, but not fibroblast, cultures containing high proportions of mutant mtDNA exhibited impaired mitochondrial translation. These observations indicate that mtDNA tRNA point mutations should be considered in the differential diagnosis of congenital myopathy. In addition they illustrate the diversity of phenotypes associated with this mutation in the same family and further highlight the association between mtDNA mutations and diabetes mellitus. 43 refs., 4 figs., 1 tab.

  18. A kinetic framework for tRNA ligase and enforcement of a 2'-phosphate requirement for ligation highlights the design logic of an RNA repair machine.

    PubMed

    Remus, Barbara S; Shuman, Stewart

    2013-05-01

    tRNA ligases are essential components of informational and stress-response pathways entailing repair of RNA breaks with 2',3'-cyclic phosphate and 5'-OH ends. Plant and fungal tRNA ligases comprise three catalytic domains. Phosphodiesterase and kinase modules heal the broken ends to generate the 3'-OH, 2'-PO₄, and 5'-PO₄ required for sealing by the ligase. We exploit RNA substrates with different termini to define rates of individual steps or subsets of steps along the repair pathway of plant ligase AtRNL. The results highlight rate-limiting transactions, how repair is affected by active-site mutations, and how mutations are bypassed by RNA alterations. We gain insights to 2'-PO₄ specificity by showing that AtRNL is deficient in transferring AMP to pRNAOH to form AppRNAOH but proficient at sealing pre-adenylylated AppRNAOH. This strategy for discriminating 2'-PO₄ versus 2'-OH ends provides a quality-control checkpoint to ensure that only purposeful RNA breaks are sealed and to avoid nonspecific "capping" of 5'-PO₄ ends.

  19. Association between allelic variation due to short tandem repeats in tRNA gene of Entamoeba histolytica and clinical phenotypes of amoebiasis.

    PubMed

    Jaiswal, Virendra; Ghoshal, Ujjala; Mittal, Balraj; Dhole, Tapan N; Ghoshal, Uday C

    2014-05-01

    Genotypes of Entamoeba histolytica (E. histolytica) may contribute clinical phenotypes of amoebiasis such as amoebic liver abscess (ALA), dysentery and asymptomatic cyst passers state. Hence, we evaluated allelic variation due to short tandem repeats (STRs) in tRNA gene of E. histolytica and clinical phenotypes of amoebiasis. Asymptomatic cyst passers (n=24), patients with dysentery (n=56) and ALA (n=107) were included. Extracted DNA from stool (dysentery, asymptomatic cyst passers) and liver aspirate was amplified using 6 E. histolytica specific tRNA-linked STRs (D-A, A-L, N-K2, R-R, S-Q, and S(TGA)-D) primers. PCR products were subjected to sequencing. Association between allelic variation and clinical phenotypes was analyzed. A total of 9 allelic variations were found in D-A, 8 in A-L, 4 in N-K2, 5 in R-R, 10 in S(TAG)-D and 7 in S-Q loci. A significant association was found between allelic variants and clinical phenotypes of amoebiasis. This study reveals that allelic variation due to short tandem repeats (STRs) in tRNA gene of E. histolytica is associated different clinical outcome of amoebiasis.

  20. Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

    PubMed Central

    French, B T; Patrick, D E; Grever, M R; Trewyn, R W

    1991-01-01

    6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells. Images PMID:1988936

  1. Active site nanospace of aminoacyl tRNA synthetase: difference between the class I and class II synthetases.

    PubMed

    Dutta, Saheb; Choudhury, Kaberi; Banik, Sindrila Dutta; Nandi, Nilashis

    2014-03-01

    The present work is aimed at understanding the origin of the difference in the molecular organization of the active site nanospaces of the class I and class II aminoacyl tRNA synthetases (aaRSs) which are tunnel-like structures. The active site encloses the cognate amino acid (AA) and the adenosine triphosphate (ATP) to carry out aminoacylation reaction. Comparison of the structures of the active site of the class I and class II (aaRSs) shows that the nanodimensional tunnels are curved in opposite directions in the two classes. We investigated the origin of this difference using quantum mechanical computation of electrostatic potential (ESP) of substrates, surrounding residues and ions, using Atoms in Molecule (AIM) Theory and charge population analysis. We show that the difference is principally due to the variation in the spatial charge distribution of ATP in the two classes which correspond to extended and bent conformations of ATP. The present computation shows that the most feasible pathway for nucleophilic attack to alphaP is oppositely directed for class I and class II aaRSs. The available crystal structures show that the cognate AA is indeed located along the channel favorable for nucleophilic attack as predicted by the ESP analysis. It is also shown that the direction of the channel changes its orientation when the orientation of ATP is changed from extended to a bent like structure. We further used the AIM theory to confirm the direction of the approach of AA in each case and the results corroborate the results from the ESP analysis. The opposite curvatures of the active site nanospaces in class I and class II aaRSs are related with the influence of the charge distributions of the extended and bent conformations of ATP, respectively. The results of the computation of electrostatic potential by successive addition of active site residues show that their roles on the reaction are similar in both classes despite the difference in the organization of the

  2. The brome mosaic virus RNA3 intergenic replication enhancer folds to mimic a tRNA TpsiC-stem loop and is modified in vivo.

    PubMed Central

    Baumstark, T; Ahlquist, P

    2001-01-01

    The genome of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, consists of three capped, messenger-sense RNAs. RNA1 and RNA2 encode viral replication proteins 1a and 2a, respectively. RNA3 encodes the 3a movement protein and the coat protein, which are essential for systemic infection in plants but dispensable for RNA3 replication in plants and yeast. A subset of the 250-base intergenic region (IGR), the replication enhancer (RE), contains all cis-acting signals necessary for a crucial, early template selection step, the 1a-dependent recruitment of RNA3 into replication. One of these signals is a motif matching the conserved box B sequence of RNA polymerase III transcripts. Using chemical modification with CMCT, kethoxal, DMS, DEPC, and lead, we probed the structure of the IGR in short, defined transcripts and in full-length RNA3 in vitro, in yeast extracts, and in whole yeast cells. Our results reveal a stable, unbranched secondary structure that is not dependent on the surrounding ORF sequences or on host factors within the cell. Functional 5' and 3' deletions that defined the minimal RE in earlier deletion studies map to the end of a common helical segment. The box B motif is presented as a hairpin loop of 7 nt closed by G:C base pairs in perfect analogy to the TpsiC-stem loop in tRNA(Asp). An adjacent U-rich internal loop, a short helix, and another pyrimidine-rich loop were significantly protected from base modifications. This same arrangement is conserved between BMV and cucumoviruses CMV, TAV, and PSV. In the BMV box B loop sequence, uridines corresponding to tRNA positions T54 and psi55 were found to be modified in yeast and plants to 5mU and pseudouridine. Together with the aminoacylated viral 3'-end, this is thus the second RNA replication signal within BMV where the virus has evolved a tRNA structural mimicry to a degree that renders it a substrate for classical tRNA modification reactions in vivo. PMID:11720293

  3. The transition from noncoded to coded protein synthesis: did coding mRNAs arise from stability-enhancing binding partners to tRNA?

    PubMed Central

    2010-01-01

    Background Understanding the origin of protein synthesis has been notoriously difficult. We have taken as a starting premise Wolf and Koonin's view that "evolution of the translation system is envisaged to occur in a compartmentalized ensemble of replicating, co-selected RNA segments, i.e., in an RNA world containing ribozymes with versatile activities". Presentation of the hypothesis We propose that coded protein synthesis arose from a noncoded process in an RNA world as a natural consequence of the accumulation of a range of early tRNAs and their serendipitous RNA binding partners. We propose that, initially, RNA molecules with 3' CCA termini that could be aminoacylated by ribozymes, together with an ancestral peptidyl transferase ribozyme, produced small peptides with random or repetitive sequences. Our concept is that the first tRNA arose in this context from the ligation of two RNA hairpins and could be similarly aminoacylated at its 3' end to become a substrate for peptidyl transfer catalyzed by the ancestral ribozyme. Within this RNA world we hypothesize that proto-mRNAs appeared first simply as serendipitous binding partners, forming complementary base pair interactions with the anticodon loops of tRNA pairs. Initially this may have enhanced stability of the paired tRNA molecules so they were held together in close proximity, better positioning the 3' CCA termini for peptidyl transfer and enhancing the rate of peptide synthesis. If there were a selective advantage for the ensemble through the peptide products synthesized, it would provide a natural pathway for the evolution of a coding system with the expansion of a cohort of different tRNAs and their binding partners. The whole process could have occurred quite unremarkably for such a profound acquisition. Testing the hypothesis It should be possible to test the different parts of our model using the isolated contemporary 50S ribosomal subunit initially, and then with RNAs transcribed in vitro together

  4. Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

    PubMed

    Meinnel, T; Mechulam, Y; Fayat, G; Blanquet, S

    1992-09-25

    The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met). PMID:1408786

  5. Virulence characteristics of Salmonella following deletion of genes encoding the tRNA modification enzymes GidA and MnmE.

    PubMed

    Shippy, Daniel C; Eakley, Nicholas M; Lauhon, Charles T; Bochsler, Philip N; Fadl, Amin A

    2013-04-01

    Salmonella is an important foodborne pathogen causing major public health problems throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division (gidA) gene significantly altered Salmonella virulence in both in vitro and in vivo models of infection. In Escherichia coli, GidA and MnmE have been shown to modify several bacterial factors by a post-transcriptional mechanism to modify tRNA. Therefore, we hypothesize that GidA and MnmE complex together to modulate virulence genes in Salmonella using a similar mechanism. To test our hypothesis, and to examine the relative contribution of GidA and MnmE in modulation of Salmonella virulence, we constructed gidA and mnmE single mutants as well as a gidA mnmE double mutant strain of Salmonella. Results from the in vitro data displayed a reduction in growth, motility, intracellular replication, and invasion of T84 intestinal epithelial cells in the mutant strains compared to the wild-type Salmonella strain. The in vivo data showed a significant attenuation of the mutant strains as indicated by the induction of inflammatory cytokines and chemokines, as well as in the severity of histopathological lesions in the liver and spleen, compared to mice infected with the wild-type strain. Also, a significant increase in the LD50 was observed in mice infected with the mutant strains, and mice immunized with the mutants were protected against a lethal dose of wild-type Salmonella. A pull-down assay indicated that Salmonella GidA and MnmE bind together, and HPLC analysis revealed that deletion of gidA and/or mnmE altered Salmonella tRNA modification. Overall, the data suggest MnmE and GidA bind together and use a post-transcriptional mechanism to modify tRNA to regulate Salmonella pathogenesis.

  6. Novel mitochondrial tRNA Leu(CUN) transition and D4Z4 partial deletion in a patient with a facioscapulohumeral phenotype.

    PubMed

    Filosto, Massimiliano; Tonin, Paola; Scarpelli, Mauro; Savio, Chiara; Greco, Francesca; Mancuso, Michelangelo; Vattemi, Gaetano; Govoni, Vittorio; Rizzuto, Nicolò; Tupler, Rossella; Tomelleri, Giuliano

    2008-03-01

    Point mutations in mtDNA-encoded tRNA genes frequently cause isolated myopathies but rarely cause the facioscapulohumeral phenotype. We report on a patient affected with chronic progressive weakness of facioscapulohumeral/peroneal muscles whose muscle biopsy showed a mitochondrial myopathy. mtDNA direct sequencing and RFLP analysis revealed a heteroplasmic transition T12313C which disrupts a conserved site in the T Psi C stem of the tRNA(Leu(CUN)) gene and fulfills the accepted criteria of pathogenicity. A partial deletion of the nuclear DNA D4Z4 region with residual repeat sizes of 25 kb was also found in the patient and in her mother. This is the first reported case of mitochondrial myopathy/facioscapulohumeral muscular dystrophy (FSHD) "double trouble". PMID:18343111

  7. Aquifex aeolicus Trm1[tRNA (m2(2)G26) methyltransferase] has a novel recognition mechanism of the substrate RNA.

    PubMed

    Awai, Takako; Takehara, Toru; Takeda, Hiroshi; Hori, Hiroyuki

    2005-01-01

    Aquifex aeolicus is a hyper-thermophilic eubacterium, which probably diverged at the earliest period from the other bacteria on the evolution of life. Therefore, analyses of RNA modification enzymes of this bacterium may supply important information in relation to the establishment of the early protein synthesis. In the previous meeting, we have reported that Aquifex aeolicus trm1 gene product possesses a tRNA (m2(2)G26) methyltransferase activity. This enzyme catalyzes the methyl-transfer from S-adenosyl-L-methionine to the semi-conserved residue, G26, via the intermediate modified base, m2G26. In this meeting, we report the A. aeolicus Trm1 large scale expression system in E. coli and the substrate RNA recognition mechanism of the enzyme.

  8. Free-Energy Landscape of Reverse tRNA Translocation through the Ribosome Analyzed by Electron Microscopy Density Maps and Molecular Dynamics Simulations

    PubMed Central

    Ishida, Hisashi; Matsumoto, Atsushi

    2014-01-01

    To understand the mechanism of reverse tRNA translocation in the ribosome, all-atom molecular dynamics simulations of the ribosome-tRNAs-mRNA-EFG complex were performed. The complex at the post-translocational state was directed towards the translocational and pre-translocational states by fitting the complex into cryo-EM density maps. Between a series of the fitting simulations, umbrella sampling simulations were performed to obtain the free-energy landscape. Multistep structural changes, such as a ratchet-like motion and rotation of the head of the small subunit were observed. The free-energy landscape showed that there were two main free-energy barriers: one between the post-translocational and intermediate states, and the other between the pre-translocational and intermediate states. The former corresponded to a clockwise rotation, which was coupled to the movement of P-tRNA over the P/E-gate made of G1338, A1339 and A790 in the small subunit. The latter corresponded to an anticlockwise rotation of the head, which was coupled to the location of the two tRNAs in the hybrid state. This indicates that the coupled motion of the head rotation and tRNA translocation plays an important role in opening and closing of the P/E-gate during the ratchet-like movement in the ribosome. Conformational change of EF-G was interpreted to be the result of the combination of the external motion by L12 around an axis passing near the sarcin-ricin loop, and internal hinge-bending motion. These motions contributed to the movement of domain IV of EF-G to maintain its interaction with A/P-tRNA. PMID:24999999

  9. Coexistence of bacterial leucyl-tRNA synthetases with archaeal tRNA binding domains that distinguish tRNALeu in the archaeal mode

    PubMed Central

    Fang, Zhi-Peng; Wang, Meng; Ruan, Zhi-Rong; Tan, Min; Liu, Ru-Juan; Zhou, Mi; Zhou, Xiao-Long; Wang, En-Duo

    2014-01-01

    Leucyl-tRNA (transfer RNA) synthetase (LeuRS) is a multi-domain enzyme, which is divided into bacterial and archaeal/eukaryotic types. In general, one specific LeuRS, the domains of which are of the same type, exists in a single cell compartment. However, some species, such as the haloalkaliphile Natrialba magadii, encode two cytoplasmic LeuRSs, NmLeuRS1 and NmLeuRS2, which are the first examples of naturally occurring chimeric enzymes with different domains of bacterial and archaeal types. Furthermore, N. magadii encodes typical archaeal tRNALeus. The tRNA recognition mode, aminoacylation and translational quality control activities of these two LeuRSs are interesting questions to be addressed. Herein, active NmLeuRS1 and NmLeuRS2 were successfully purified after gene expression in Escherichia coli. Under the optimized aminoacylation conditions, we discovered that they distinguished cognate NmtRNALeu in the archaeal mode, whereas the N-terminal region was of the bacterial type. However, NmLeuRS1 exhibited much higher aminoacylation and editing activity than NmLeuRS2, suggesting that NmLeuRS1 is more likely to generate Leu-tRNALeu for protein biosynthesis. Moreover, using NmLeuRS1 as a model, we demonstrated misactivation of several non-cognate amino acids, and accuracy of protein synthesis was maintained mainly via post-transfer editing. This comprehensive study of the NmLeuRS/tRNALeu system provides a detailed understanding of the coevolution of aminoacyl-tRNA synthetases and tRNA. PMID:24500203

  10. TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies

    PubMed Central

    Powell, Christopher A.; Kopajtich, Robert; D’Souza, Aaron R.; Rorbach, Joanna; Kremer, Laura S.; Husain, Ralf A.; Dallabona, Cristina; Donnini, Claudia; Alston, Charlotte L.; Griffin, Helen; Pyle, Angela; Chinnery, Patrick F.; Strom, Tim M.; Meitinger, Thomas; Rodenburg, Richard J.; Schottmann, Gudrun; Schuelke, Markus; Romain, Nadine; Haller, Ronald G.; Ferrero, Ileana; Haack, Tobias B.; Taylor, Robert W.; Prokisch, Holger; Minczuk, Michal

    2015-01-01

    Deficiencies in respiratory-chain complexes lead to a variety of clinical phenotypes resulting from inadequate energy production by the mitochondrial oxidative phosphorylation system. Defective expression of mtDNA-encoded genes, caused by mutations in either the mitochondrial or nuclear genome, represents a rapidly growing group of human disorders. By whole-exome sequencing, we identified two unrelated individuals carrying compound heterozygous variants in TRMT5 (tRNA methyltransferase 5). TRMT5 encodes a mitochondrial protein with strong homology to members of the class I-like methyltransferase superfamily. Both affected individuals presented with lactic acidosis and evidence of multiple mitochondrial respiratory-chain-complex deficiencies in skeletal muscle, although the clinical presentation of the two affected subjects was remarkably different; one presented in childhood with failure to thrive and hypertrophic cardiomyopathy, and the other was an adult with a life-long history of exercise intolerance. Mutations in TRMT5 were associated with the hypomodification of a guanosine residue at position 37 (G37) of mitochondrial tRNA; this hypomodification was particularly prominent in skeletal muscle. Deficiency of the G37 modification was also detected in human cells subjected to TRMT5 RNAi. The pathogenicity of the detected variants was further confirmed in a heterologous yeast model and by the rescue of the molecular phenotype after re-expression of wild-type TRMT5 cDNA in cells derived from the affected individuals. Our study highlights the importance of post-transcriptional modification of mitochondrial tRNAs for faithful mitochondrial function. PMID:26189817

  11. Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

    PubMed

    Jones, Christopher P; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

    2013-02-01

    The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

  12. tRNA modification by S-adenosylmethionine:tRNA ribosyltransferase-isomerase. Assay development and characterization of the recombinant enzyme.

    PubMed

    Van Lanen, Steven G; Kinzie, Sylvia Daoud; Matthieu, Sharlene; Link, Todd; Culp, Jeff; Iwata-Reuyl, Dirk

    2003-03-21

    The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA(Tyr) in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-(14)C]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg(2+) and Mn(2+) in millimolar concentrations. The steady-state kinetic parameters were determined to be K(m)(AdoMet) = 101.4 microm, K(m)(tRNA) = 1.5 microm, and k(cat) = 2.5 min(-1). A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K(m)(RNA) = 37.7 microm and k(cat) = 14.7 min(-1)), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA. PMID:12533518

  13. A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

    PubMed Central

    Desai, Kevin K.; Cheng, Chin L.; Bingman, Craig A.; Phillips, George N.; Raines, Ronald T.

    2014-01-01

    Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3′-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon. PMID:24435797

  14. The initiator tRNA acceptance assay as a short-term test for carcinogens. 2. Results with ten compounds selected by the International Programme on Chemical Safety for the evaluation of short-term tests for carcinogens.

    PubMed

    Hradec, J; Spiegelhalder, B; Preussmann, R

    1988-05-01

    Eight carcinogenic and two non-carcinogenic compounds that are difficult to detect by short-term tests (acrylonitrile, benzene, benzoin, caprolactam, diethylhexylphtalate, diethylstilbestrol, hexamethylphosphoramide, phenobarbital, safrole and o-toluidine) were tested independently in Prague and in Heidelberg by the newly developed initiator tRNA acceptance assay. Seven out of eight tested carcinogens gave a positive response in this assay, only safrole showed a false negativity in both laboratories. Both non-carcinogenic compounds, benzoin and caprolactam, exhibited no activity. An absolute qualitative agreement was found with all compounds tested between the results of both laboratories. With the exception only of phenobarbital (intermediate activity in Prague and low in Heidelberg) the quantitative results obtained in both laboratories were comparable. The initiator tRNA acceptance assay thus appears to be a reliable short-term test for carcinogenicity with good reproducibility.

  15. Stabilization of the tertiary structure of yeast phenylalanine tRNA by [Co(NH3)6]3+. X-ray evidence for hydrogen bonding to pairs of guanine bases in the major groove.

    PubMed

    Hingerty, B E; Brown, R S; Klug, A

    1982-04-26

    The sites of three [Co(NH3)6]3+ ions bound to the phenylalanine tRNA of yeast have been determined by X-ray diffraction analysis. [Co(NH3)6]3+ binds to purine-purine sequences in yeast tRNA Phe. It is different from the binding fo Co2+, which binds to the base and phosphate of residue G15. There are no direct metal-nucleotide bonds, although hydrogen bonding of the coordinated ammines to double-helical guanylguanosine sequences in the major groove and to phosphate oxygen in neighboring polynucleotide strands increases the stability of the structure. Hydrogen-bonding appears to be via cis ammine ligands to N(7) and O(6) positions of adjacent purine bases.

  16. The archaeal COG1901/DUF358 SPOUT-methyltransferase members, together with pseudouridine synthase Pus10, catalyze the formation of 1-methylpseudouridine at position 54 of tRNA.

    PubMed

    Chatterjee, Kunal; Blaby, Ian K; Thiaville, Patrick C; Majumder, Mrinmoyee; Grosjean, Henri; Yuan, Y Adam; Gupta, Ramesh; de Crécy-Lagard, Valérie

    2012-03-01

    The methylation of pseudouridine (Ψ) at position 54 of tRNA, producing m(1)Ψ, is a hallmark of many archaeal species, but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m(1)Ψ was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m(1)Ψ minus phenotype of the ΔHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TΨ-arm (17-mer) fragments as substrates, the sequential pathway of m(1)Ψ54 formation in Archaea was reconstituted. The methylation reaction is AdoMet dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TΨ-loop. The presence of Ψ55 allowed the efficient conversion of Ψ54 to m(1)Ψ54, whereas in the presence of C55, the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins.

  17. A survey of green plant tRNA 3'-end processing enzyme tRNase Zs, homologs of the candidate prostate cancer susceptibility protein ELAC2

    PubMed Central

    2011-01-01

    Background tRNase Z removes the 3'-trailer sequences from precursor tRNAs, which is an essential step preceding the addition of the CCA sequence. tRNase Z exists in the short (tRNase ZS) and long (tRNase ZL) forms. Based on the sequence characteristics, they can be divided into two major types: bacterial-type tRNase ZS and eukaryotic-type tRNase ZL, and one minor type, Thermotoga maritima (TM)-type tRNase ZS. The number of tRNase Zs is highly variable, with the largest number being identified experimentally in the flowering plant Arabidopsis thaliana. It is unknown whether multiple tRNase Zs found in A. thaliana is common to the plant kingdom. Also unknown is the extent of sequence and structural conservation among tRNase Zs from the plant kingdom. Results We report the identification and analysis of candidate tRNase Zs in 27 fully sequenced genomes of green plants, the great majority of which are flowering plants. It appears that green plants contain multiple distinct tRNase Zs predicted to reside in different subcellular compartments. Furthermore, while the bacterial-type tRNase ZSs are present only in basal land plants and green algae, the TM-type tRNase ZSs are widespread in green plants. The protein sequences of the TM-type tRNase ZSs identified in green plants are similar to those of the bacterial-type tRNase ZSs but have distinct features, including the TM-type flexible arm, the variant catalytic HEAT and HST motifs, and a lack of the PxKxRN motif involved in CCA anti-determination (inhibition of tRNase Z activity by CCA), which prevents tRNase Z cleavage of mature tRNAs. Examination of flowering plant chloroplast tRNA genes reveals that many of these genes encode partial CCA sequences. Based on our results and previous studies, we predict that the plant TM-type tRNase ZSs may not recognize the CCA sequence as an anti-determinant. Conclusions Our findings substantially expand the current repertoire of the TM-type tRNase ZSs and hint at the possibility that

  18. Peptidyl-CCA deacylation on the ribosome promoted by induced fit and the O3′-hydroxyl group of A76 of the unacylated A-site tRNA

    SciTech Connect

    Simonović, Miljan; Steitz, Thomas A.

    2008-11-24

    The last step in ribosome-catalyzed protein synthesis is the hydrolytic release of the newly formed polypeptide from the P-site bound tRNA. Hydrolysis of the ester link of the peptidyl-tRNA is stimulated normally by the binding of release factors (RFs). However, an unacylated tRNA or just CCA binding to the ribosomal A site can also stimulate deacylation under some nonphysiological conditions. Although the sequence of events is well described by biochemical studies, the structural basis of the mechanism underlying this process is not well understood. Two new structures of the large ribosomal subunit of Haloarcula marismortui complexed with a peptidyl-tRNA analog in the P site and two oligonucleotide mimics of unacylated tRNA, CCA and CA, in the A site show that the binding of either CA or CCA induces a very similar conformational change in the peptidyl-transferase center as induced by aminoacyl-CCA. However, only CCA positions a water molecule appropriately to attack the carbonyl carbon of the peptidyl-tRNA and stabilizes the proper orientation of the ester link for hydrolysis. We, thus, conclude that both the ability of the O3'-hydroxyl group of the A-site A76 to position the water and the A-site CCA induced conformational change of the PTC are critical for the catalysis of the deacylation of the peptidyl-tRNA by CCA, and perhaps, an analogous mechanism is used by RFs.

  19. Selection of aminoacyl-tRNAs at sense codons: the size of the tRNA variable loop determines whether the immediate 3' nucleotide to the codon has a context effect.

    PubMed Central

    Curran, J F; Poole, E S; Tate, W P; Gross, B L

    1995-01-01

    Codon context can affect translational efficiency by several molecular mechanisms. The base stacking interactions between a codon-anticodon complex and the neighboring nucleotide immediately 3' can facilitate translation by amber suppressors and the tRNA structure is also known to modulate the sensitivity to context. In this study the relative rates of aminoacyl-tRNA selection were measured at four sense codons (UGG, CUC, UUC and UCA), in all four 3' nucleotide contexts, through direct competition with a programmed frameshift at a site derived from the release factor 2 gene. Two codons (UGG and UUC) are read by tRNAs with small variable regions and their rates of aminoacyl-tRNA selection correlated with the potential base stacking strength of the 3' neighboring nucleotide. The other two codons (CUC and UCA) are read by tRNAs with large variable regions and the rate of selection of the aminoacyl-tRNAs in these cases varied little among the four contexts. Re-examination of published data on amber suppression also revealed an inverse correlation between context sensitivity and the size of the variable region. Collectively the data suggest that a large variable loop in a tRNA decreases the influence of the 3' context on tRNA selection, probably by strengthening tRNA-ribosomal interactions. PMID:7479072

  20. Archaeal Tuc1/Ncs6 Homolog Required for Wobble Uridine tRNA Thiolation Is Associated with Ubiquitin-Proteasome, Translation, and RNA Processing System Homologs

    PubMed Central

    Chavarria, Nikita E.; Hwang, Sungmin; Cao, Shiyun; Fu, Xian; Holman, Mary; Elbanna, Dina; Rodriguez, Suzanne; Arrington, Deanna; Englert, Markus; Uthandi, Sivakumar; Söll, Dieter; Maupin-Furlow, Julie A.

    2014-01-01

    While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNALysUUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNALysUUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems. PMID:24906001

  1. Long-Range Structural Effects of a Charcot-Marie-Tooth Disease-Causing Mutation in Human Glycyl-TRNA Synthetase

    SciTech Connect

    Xie, W.; Nangle, L.A.; Zhang, W.; Schimmel, P.; Yang, X.-L.

    2009-06-04

    Functional expansion of specific tRNA synthetases in higher organisms is well documented. These additional functions may explain why dominant mutations in glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase cause Charcot-Marie-Tooth (CMT) disease, the most common heritable disease of the peripheral nervous system. At least 10 disease-causing mutant alleles of GlyRS have been annotated. These mutations scatter broadly across the primary sequence and have no apparent unifying connection. Here we report the structure of wild type and a CMT-causing mutant (G526R) of homodimeric human GlyRS. The mutation is at the site for synthesis of glycyl-adenylate, but the rest of the two structures are closely similar. Significantly, the mutant form diffracts to a higher resolution and has a greater dimer interface. The extra dimer interactions are located {approx}30 {angstrom} away from the G526R mutation. Direct experiments confirm the tighter dimer interaction of the G526R protein. The results suggest the possible importance of subtle, long-range structural effects of CMT-causing mutations at the dimer interface. From analysis of a third crystal, an appended motif, found in higher eukaryote GlyRSs, seems not to have a role in these long-range effects.

  2. Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth disease

    SciTech Connect

    Xie, Wei; Schimmel, Paul; Yang, Xiang-Lei

    2006-12-01

    Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth Disease. Glycyl-tRNA synthetase (GlyRS) is one of a group of enzymes that catalyze the synthesis of aminoacyl-tRNAs for translation. Mutations of human and mouse GlyRSs are causally associated with Charcot–Marie–Tooth disease, the most common genetic disorder of the peripheral nervous system. As the first step towards a structure–function analysis of this disease, native human GlyRS was expressed, purified and crystallized. The crystal belonged to space group P4{sub 3}2{sub 1}2 or its enantiomorphic space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 91.74, c = 247.18 Å, and diffracted X-rays to 3.0 Å resolution. The asymmetric unit contained one GlyRS molecule and had a solvent content of 69%.

  3. Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

    PubMed

    Fabrizio, Paola; Hoon, Shawn; Shamalnasab, Mehrnaz; Galbani, Abdulaye; Wei, Min; Giaever, Guri; Nislow, Corey; Longo, Valter D

    2010-07-15

    The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

  4. Identification of an amber nonsense mutation in the rosy516 gene by germline transformation of an amber suppressor tRNA gene.

    PubMed Central

    Doerig, R E; Suter, B; Gray, M; Kubli, E

    1988-01-01

    Seven xanthine dehydrogenase and cross-reacting material negative Drosophila melanogaster rosy stocks were screened for amber and ochre nonsense mutations. Amber and ochre nonsense suppressors were created by site-directed mutagenesis starting from a wild-type tRNA(Tyr) gene. The suppressor tRNA genes were subcloned into a pUChsneo transformation vector providing heat-shock controlled neomycin resistance. The seven rosy stocks were germline transformed with amber and ochre tDNA(Tyr), and the G1 generation was screened for Geneticin resistance. Surviving rosy516 flies transformed with the amber suppressor showed an eye colour intermediate between the original ry516 stock and the wild-type, suggesting that ry516 is an amber nonsense mutant. This was confirmed by sequencing the relevant part of the ry516 gene; the analysis revealed a C-to-T transition in a CAG glutamine codon at nucleotide 1522 of the wild-type rosy gene. Images PMID:3142765

  5. Near-UV stress in salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response

    SciTech Connect

    Kramer, G.F.; Baker, J.C.; Ames, B.N.

    1988-05-01

    We have examined the role of 4-thiouridine in the responses of Salmonella typhimurium to near-UV irradiation. Mutants lacking 4-thiouridine (nuv) and mutants defective in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (relA) were found to be sensitive to killing by near-UV. Near-UV induced the synthesis of a set of proteins that were not induced in the nuv mutant. Some of these proteins were identified as oxidative defense proteins, and others were identified as ppGpp-inducible proteins. Over 100-fold increases in ApppGpp (adenoisine 5', 5'''-triphosphoguanosine-3'''-diphosphate, the adenylylated form of ppGpp) were observed in wild-type cells after near-UV irradiation but not in the 4-thiouridine-deficient mutant. These data support a model in which ppGpp and ApppGpp, a dinucleotide proposed to be synthesized by tRNA-aminoacyl synthetases as a response to the cross-linking of 4-thiouridine in tRNA by near-UV, induce the synthesis of proteins necessary for resistance to near-UV irradiation.

  6. Near-UV stress in Salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response.

    PubMed

    Kramer, G F; Baker, J C; Ames, B N

    1988-05-01

    We have examined the role of 4-thiouridine in the responses of Salmonella typhimurium to near-UV irradiation. Mutants lacking 4-thiouridine (nuv) and mutants defective in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (relA) were found to be sensitive to killing by near-UV. Near-UV induced the synthesis of a set of proteins that were not induced in the nuv mutant. Some of these proteins were identified as oxidative defense proteins, and others were identified as ppGpp-inducible proteins. Over 100-fold increases in ApppGpp (adenosine 5', 5"'-triphosphoguanosine-3"'-diphosphate, the adenylylated form of ppGpp) were observed in wild-type cells after near-UV irradiation but not in the 4-thiouridine-deficient mutant. These data support a model in which ppGpp and ApppGpp, a dinucleotide proposed to be synthesized by tRNA-aminoacyl synthetases as a response to the cross-linking of 4-thiouridine in tRNA by near-UV, induce the synthesis of proteins necessary for resistance to near-UV irradiation.

  7. Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit.

    PubMed

    Pan, Hu; Agarwalla, Sanjay; Moustakas, Demetri T; Finer-Moore, Janet; Stroud, Robert M

    2003-10-28

    RNA pseudouridine synthase, TruB, catalyzes pseudouridine formation at U55 in tRNA. This posttranscriptional modification is almost universally conserved and occurs in the T arm of most tRNAs. We determined the crystal structure of Escherichia coli TruB apo enzyme, as well as the structure of Thermotoga maritima TruB in complex with RNA. Comparison of the RNA-free and -bound forms of TruB reveals that this enzyme undergoes significant conformational changes on binding to its substrate. These conformational changes include the ordering of the "thumb loop," which binds right into the RNA hairpin loop, and a 10 degree hinge movement of the C-terminal domain. Along with the result of docking experiments performed on apo TruB, we conclude that TruB recognizes its RNA substrate through a combination of rigid docking and induced fit, with TruB first rigidly binding to its target and then maximizing the interaction by induced fit.

  8. Expression of Selenoproteins Is Maintained in Mice Carrying Mutations in SECp43, the tRNA Selenocysteine 1 Associated Protein (Trnau1ap)

    PubMed Central

    Carlson, Bradley A.; Fradejas, Noelia; Günter, Paul; Braun, Doreen; Southon, Eileen; Tessarollo, Lino; Hatfield, Dolph L.; Schweizer, Ulrich

    2015-01-01

    Selenocysteine tRNA 1 associated protein (Trnau1ap) has been characterized as a tRNA[Ser]Sec-binding protein of 43 kDa, hence initially named SECp43. Previous studies reported its presence in complexes containing tRNA[Ser]Sec implying a role of SECp43 as a co-factor in selenoprotein expression. We generated two conditionally mutant mouse models targeting exons 3+4 and exons 7+8 eliminating parts of the first RNA recognition motif or of the tyrosine-rich domain, respectively. Constitutive inactivation of exons 3+4 of SECp43 apparently did not affect the mice or selenoprotein expression in several organs. Constitutive deletion of exons 7+8 was embryonic lethal. We therefore generated hepatocyte-specific Secp43 knockout mice and characterized selenoprotein expression in livers of mutant mice. We found no significant changes in the levels of 75Se-labelled hepatic proteins, selenoprotein levels as determined by Western blot analysis, enzymatic activity or selenoprotein mRNA abundance. The methylation pattern of tRNA[Ser]Sec remained unchanged. Truncated Secp43 Δ7,8mRNA increased in Secp43-mutant livers suggesting auto-regulation of Secp43 mRNA abundance. We found no signs of liver damage in Secp433-mutant mice, but neuron-specific deletion of exons 7+8 impaired motor performance, while not affecting cerebral selenoprotein expression or cerebellar development. These findings suggest that the targeted domains in the SECp43 protein are not essential for selenoprotein biosynthesis in hepatocytes and neurons. Whether the remaining second RNA recognition motif plays a role in selenoprotein biosynthesis and which other cellular process depends on SECp43 remains to be determined. PMID:26043259

  9. A Novel Pathogenicity Island Integrated Adjacent to the thrW tRNA Gene of Avian Pathogenic Escherichia coli Encodes a Vacuolating Autotransporter Toxin

    PubMed Central

    Parreira, V. R.; Gyles, C. L.

    2003-01-01

    We report the complete nucleotide sequence and genetic organization of the Vat-encoding pathogenicity island (PAI) of avian pathogenic Escherichia coli strain Ec222. The 22,139-bp PAI is situated adjacent to the 3′ terminus of the thrW tRNA gene, has a G+C content of 41.2%, and includes a bacteriophage SfII integrase gene, mobile genetic elements, two open reading frames with products exhibiting sequence similarity to known proteins, and several other open reading frames of unknown function. The PAI encodes an autotransporter protein, Vat (vacuolating autotransporter toxin), which induces the formation of intracellular vacuoles resulting in cytotoxic effects similar to those caused by the VacA toxin from Helicobacter pylori. The predicted 148.3-kDa protein product possesses the three domains that are typical of serine protease autotransporters of Enterobacteriaceae: an N-terminal signal sequence of 55 amino acids, a 111.8-kDa passenger domain containing a modified serine protease site (ATSGSG), and a C-terminal outer membrane translocator of 30.5 kDa. Vat has 75% protein homology with the hemagglutinin Tsh, an autotransporter of avian pathogenic E. coli. A vat deletion mutant of Ec222 showed no virulence in respiratory and cellulitis infection models of disease in broiler chickens. We conclude that the newly described PAI and Vat may be involved in the pathogenicity of avian septicemic E. coli strain Ec222 and other avian pathogenic E. coli strains. PMID:12933851

  10. The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis

    PubMed Central

    Clarke, Cassie J.; Berg, Tracy J.; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L.; Vermeulen, Peter B.; Foo, Shane; Kostaras, Eleftherios; Jones, J. Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R.; Norman, Jim C.

    2016-01-01

    Summary Expression of the initiator methionine tRNA (tRNAiMet) is deregulated in cancer. Despite this fact, it is not currently known how tRNAiMet expression levels influence tumor progression. We have found that tRNAiMet expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAiMet in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAiMet contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAiMet gene (2+tRNAiMet mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAiMet mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAiMet mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAiMet-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAiMet-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAiMet mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAiMet levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. PMID:26948875

  11. The complete mitochondrial genome sequence of the hydrothermal vent galatheid crab Shinkaia crosnieri (Crustacea: Decapoda: Anomura): A novel arrangement and incomplete tRNA suite

    PubMed Central

    Yang, Jin-Shu; Yang, Wei-Jun

    2008-01-01

    Background Metazoan mitochondrial genomes usually consist of the same 37 genes. Such genes contain useful information for phylogenetic analyses and evolution modelling. Although complete mitochondrial genomes have been determined for over 1,000 animals to date, hydrothermal vent species have, thus far, remained excluded due to the scarcity of collected specimens. Results The mitochondrial genome of the hydrothermal vent galatheid crab Shinkaia crosnieri is 15,182 bp in length, and is composed of 13 protein-coding genes, two ribosomal RNA genes and only 18 transfer RNA genes. The total AT content of the genome, as is typical for decapods, is 72.9%. We identified a non-coding control region of 327 bp according to its location and AT-richness. This is the smallest control region discovered in crustaceans so far. A mechanism of cytoplasmic tRNA import was addressed to compensate for the four missing tRNAs. The S. crosnieri mitogenome exhibits a novel arrangement of mitochondrial genes. We investigated the mitochondrial gene orders and found that at least six rearrangements from the ancestral pancrustacean (crustacean + hexapod) pattern have happened successively. The codon usage, nucleotide composition and bias show no substantial difference with other decapods. Phylogenetic analyses using the concatenated nucleotide and amino acid sequences of the 13 protein-coding genes prove consistent with the previous classification based upon their morphology. Conclusion The present study will supply considerable data of use for both genomic and evolutionary research on hydrothermal vent ecosystems. The mitochondrial genetic characteristics of decapods are sustained in this case of S. crosnieri despite the absence of several tRNAs and a number of dramatic rearrangements. Our results may provide evidence for the immigrating hypothesis about how vent species originate. PMID:18510775

  12. The effect of cold on serum thyroid hormones and hepatic 5 prime mono-deiodinase activity

    SciTech Connect

    Hesslink, R.L. Jr.; Quesada, M.; D'Alesandro, M.; Homer, L.D.; Reed, J.L.; Christopherson, R.; Young, B.A. Univ. of Alberta, Edmonton )

    1991-03-11

    Cold exposed swine have an increases serum concentration of triiodothyronine (T{sub 3}) and increased T{sub 3} production rate. It is thought that hepatic thyroxine (T{sub 4}) deiodination (5DI) contributes to circulating T{sub 3} concentrations. The authors investigated the effects of cold exposure (14 days) on energy intake, serum free T{sub 3} (FT{sub 3}) and free T{sub 4} (FT{sub 4}) levels; and 5DI in 5-month boars. Hepatic 5DI activity was determined by measuring the {sup 125}I generated from trace amounts of {sup 125}I T{sub 4}. FT{sub 3} and FT{sub 4} were assayed by RIA. Swine were housed in either 20C (control; n = 5) or 4C (cold; n = 7) chambers and given food ad libitum. Cold exposure increased energy intake by 42%. The increase (93%) in hepatic 5DI V{sub max} after cold exposure parallels the increase in whole animal T{sub 3} production and may account for FT{sub 3} values found after cold exposure.

  13. Photodegradation of 2,2{prime},5,5{prime}-tetrachlorobiphenyl in hexane

    SciTech Connect

    Miao, X.S.; Chu, S.G.; Xu, X.B.

    1996-04-01

    Polychlorinated biphenyls (PCBs) are of continuing concern due to their ubiquitous occurrence in the environment and potential health impacts on the mankind. For the environmental degradation of PCBs, photolysis may be one of the major pathways regardless of their chemical stabilities. Previous studies noted that PCBs degrade mainly with stepwise dechlorination in both aqueous and organic media. When ortho and other positions are simultaneously substituted by chlorines, it is the ortho chlorines that are lost preferentially. The photchemical behavior varies for different PCB cogeners, which may have different pathways of photodecomposition. In this study, trace amounts of tetrachlorobiphenyl was exposed in hexane under xeonon lamp to study its pathway of photodegradation. 7 refs., 2 figs.

  14. 5 prime -nucleotidase activity in a eutrophic lake and an oligotrophic lake

    SciTech Connect

    Cotner, J.B. Jr.; Wetzel, R.G. )

    1991-05-01

    Differences in enzymatic hydrolysis of dissolved organic phosphorus and subsequent phosphorus uptake were compared by using dual-labeled ({gamma}{sup {minus}32}P and 2{sup {minus}3}H) ATP in oligotrophic Lake Michigan and a moderately eutrophic lake in southeastern Michigan. More than 50% of the phosphate that was hydrolyzed was immediately taken up into bacterium-sized particles in the eutrophic lake and at a near-shore site in Lake Michigan. Less than 50% of the hydrolyzed phosphate was taken up into bacterium-sized particles at an offshore site in Lake Michigan. It is hypothesized that differences in size-fractionated uptake were the result of greater phosphorus utilization capacity in bacteria in habitats where loading of organic carbon is greater. Substantial isotope dilution of labeled phosphate uptake by unlabeled phosphate occurred, which implied that the phosphate was hydrolyzed extracellularly in both systems. Comparable nucleotidase activities were measured in the eutrophic lake and Lake Michigan, but the significance of the phosphate regenerated relative to particulate phosphorus pools was an order of magnitude greater in Lake Michigan. Seventy percent of the nucleotidase activity was inhibited by 100 {mu}M phosphate in the eutrophic lake, which suggest that most hydrolysis was by phosphatase. Therefore, nucleotidase activity may be more important to phosphorus regeneration in oligotrophic habitats than phosphatase activity.

  15. Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter.

    PubMed

    Wongsrikeao, Pimprapar; Sutou, Shizuyo; Kunishi, Miho; Dong, Ya Juan; Bai, Xuejin; Otoi, Takeshige

    2011-01-01

    By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.

  16. Characterisation of multidrug-resistant Salmonella Typhimurium 4,[5],12:i:- DT193 strains carrying a novel genomic island adjacent to the thrW tRNA locus.

    PubMed

    Trüpschuch, Sandra; Laverde Gomez, Jenny A; Ediberidze, Ia; Flieger, Antje; Rabsch, Wolfgang

    2010-06-01

    In 2006, monophasic, multidrug-resistant Salmonella enterica spp. enterica serovar 4,[5],12:i:- strains appeared as a novel serotype in Germany, associated with large diffuse outbreaks and increased need for hospitalisation. The emerging 4,[5],12:i:- strains isolated from patients in Germany belong mainly to phage type DT193 according to the Anderson phage typing scheme for S. Typhimurium (STM) and exhibit at least a tetra-drug resistance. The strains have been shown to harbour STM-specific Gifsy-1, Gifsy-2, and ST64B prophages. Furthermore, the extensive sequence similarity of the tRNA regions between one characterised 4,[5],12:i:- phage type DT193 and the S. Typhimurium LT2 strain as well as the STM-specific position of an IS200 element within the fliA-fliB intergenic region (Echeita et al., 2001) prompted us to classify them as a monophasic variant of S. Typhimurium. In 2008, the monophasic variant represented 42.2% of all S. Typhimurium isolates from human analysed at the National Reference Centre. Searching for insertions in tRNA sites resulted in the detection of an 18.4-kb fragment adjacent to the thrW tRNA locus, exhibiting a lower G+C content compared to the LT2 genome. Sequence analysis identified 17 potential ORFs. Some of them showed high similarity to enterobacterial phage sequences and sequences from Shigella boydii, Sh. dysenteriae, avian pathogenic Escherichia coli and other Escherichia spp. The biological function of this novel island with respect to virulence properties and metabolic functions is under investigation.

  17. Biomass turnover time in terrestrial ecosystems halved by land use

    NASA Astrophysics Data System (ADS)

    Erb, Karl-Heinz; Fetzel, Tamara; Plutzar, Christoph; Kastner, Thomas; Lauk, Christian; Mayer, Andreas; Niedertscheider, Maria; Körner, Christian; Haberl, Helmut

    2016-09-01

    The terrestrial carbon cycle is not well quantified. Biomass turnover time is a crucial parameter in the global carbon cycle, and contributes to the feedback between the terrestrial carbon cycle and climate. Biomass turnover time varies substantially in time and space, but its determinants are not well known, making predictions of future global carbon cycle dynamics uncertain. Land use--the sum of activities that aim at enhancing terrestrial ecosystem services--alters plant growth and reduces biomass stocks, and is hence expected to affect biomass turnover. Here we explore land-use-induced alterations of biomass turnover at the global scale by comparing the biomass turnover of the actual vegetation with that of a hypothetical vegetation state with no land use under current climate conditions. We find that, in the global average, biomass turnover is 1.9 times faster with land use. This acceleration affects all biomes roughly equally, but with large differences between land-use types. Land conversion, for example from forests to agricultural fields, is responsible for 59% of the acceleration; the use of forests and natural grazing land accounts for 26% and 15% respectively. Reductions in biomass stocks are partly compensated by reductions in net primary productivity. We conclude that land use significantly and systematically affects the fundamental trade-off between carbon turnover and carbon stocks.

  18. 7 CFR 51.1437 - Size classifications for halves.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... weight of half-kernels after all pieces, particles and dust, shell, center wall, and foreign material... example, “400” or “600-700”. (c) Tolerance for count per pound: In order to allow for variations incident... pound is specified, the actual count per pound may vary not more than 5 percent from the...

  19. 7 CFR 51.1437 - Size classifications for halves.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... weight of half-kernels after all pieces, particles and dust, shell, center wall, and foreign material... example, “400” or “600-700”. (c) Tolerance for count per pound: In order to allow for variations incident... pound is specified, the actual count per pound may vary not more than 5 percent from the...

  20. 7 CFR 51.1437 - Size classifications for halves.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... weight of half-kernels after all pieces, particles and dust, shell, center wall, and foreign material... example, “400” or “600-700”. (c) Tolerance for count per pound: In order to allow for variations incident... pound is specified, the actual count per pound may vary not more than 5 percent from the...