Nascent Peptide-Mediated Translation Elongation Arrest of Arabidopsis thaliana CGS1 mRNA Occurs Autonomously
Hitoshi OnouchiY. HaraguchiM. NakamotoDaizo KawasakiY. Nagami-YamashitaKatsunori MurotaA. Kezuka-HosomiYukako ChibaSatoshi Naito
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The Arabidopsis thaliana CGS1 gene encodes cystathionine γ-synthase, the first committed enzyme of methionine biosynthesis in higher plants. Expression of CGS1 is feedback-regulated at the step of mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). A short stretch of amino acid sequence, termed the MTO1 region, encoded within the first exon of CGS1 itself acts in cis in the regulation. In vitro analyses using wheat germ extract (WGE) revealed that AdoMet induces temporal translation arrest of CGS1 mRNA prior to mRNA degradation. This translational pausing occurs immediately downstream of the MTO1 region and is mediated by the nascent MTO1 peptide. In order to elucidate further the nature of this unique regulatory mechanism, we have examined whether a non-plant system also contains the post-transcriptional regulation activity. Despite the fact that mammals do not carry cystathionine γ-synthase, AdoMet was able to induce the MTO1 sequence-dependent translation elongation arrest in rabbit reticulocyte lysate (RRL) in a similar manner to that observed in WGE. This result suggests that MTO1 peptide-mediated translation arrest does not require a plant-specific factor and rather most probably occurs via a direct interaction between the nascent MTO1 peptide and the ribosome that has translated it. In contrast, decay intermediates of CGS1 mRNA normally observed upon induction of CGS1 mRNA decay in plant systems were not detected in RRL, raising the possibility that CGS1 mRNA degradation involves a plant-specific mechanism.In eukaryotic cells, mRNA molecules are coated with numerous RNA-binding proteins and so exist in ribonucleoproteins (mRNPs). The proteins associated with the mRNA regulate the fate of mRNA, including its localization, translation and decay. Before activation of translation, the mRNA does not display any template functions-it is masked. The coordinated activity of certain RNA-binding proteins determines the future fate of each mRNA individually. In embryo development, the temporal and spatial regulation of translation can cause a situation when the mRNA and the encoded protein are localized in different compartments and so the differentiation of the cells can be determined. The fundamentals of regulation of the mRNAs fate and functioning in nerves are similar to those already described for oo- and embryogenesis. Disorders in the mRNA masking and demasking result in the emergence of various diseases, in particular cancers and neuro-degenerative diseases.
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The germination and responses of Arabidopsis thaliana to conventional solvents were studied with agar-solution under artificial conditions. Arabidopsis thaliana and several targets were assayed for their responses to 2 commercial herbicide standards and 7 commercial herbicide products, and screened on 1 880 microbial extracts. The results showed that Arabidopsis thaliana was stable,highly efficient, simple and sensitive for screening lead compounds. The screening method with Arabidopsis thaliana could offer the multiple sieves to ensure that low active compounds would not be missed. Arabidopsis thaliana is one of the top choices of dicotyledons for screening lead compounds of herbicide.
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Polyuridylic-acid-directed polyphenylalanine [poly (U)-directed poly (Phe)] synthesis by Staphylococcus aureus ribosomes (washed in 1.0 M NH4Cl) increased linearly with increasing amount of poly (U) : the synthesis by washed staphylococcal ribosomes in the presence of about 800μg poly (U)/ml reached the same level as that by ribosomes of Escherichia coli Q13 at the optimum poly (U) concentration (about 40μg/ml). Binding of either 70S ribosomes or 30S ribosomal subunits of S. aureus U9 to poly (U) was less than that of E. coli Q13. Sedimentation analysis in a sucrose gradient showed that the amount of the heavy complex produced by interaction of poly (U) and ribosomes of S. aureus U9 was small at a high poly (U) concentration (1750 μg/ml), whereas that of E. coli Q13 was large even at 500 μg poly (U)/ml. These results suggest that low ability of the washed S. aureus ribosomes to synthesize poly (Phe) chiefly results from poor affinity of ribosomes for poly (U), since no ribonuclease activity could be found in the washed ribosomes.
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The quantitative interaction of [G-3H]gougerotin with Escherichia coli and Saccharomyces cerevisiae ribosomes is studied in this contribution. While E. coli ribosomes show an homogeneous single-site interaction with the antibiotic (dissociation constant = 1.5 μM), S. cerevisiae ribosomes are heterogeneous for [G-3H]gougerotin binding: there is a single-site interaction with dissociation constant = 0.9 μM in 28% of the ribosomes and another single binding site with dissociation constant = 12 μM in 58% of the ribosomes. Antibiotic interaction takes place in all cases with the larger ribosomal subunit. Gougerotin binding is affected by the nature of the ribosomal preparation. Thus, gougerotin has a much stronger affinity for E. coli washed ribosomes than for ribosomes reconstituted from their subunits. This difference is reduced when tRNAphe is prebound, in the presence of poly(U), to the reconstituted ribosomes. Gougerotin is also able to detect differences between the poly(U) · ribosome · Ac-Phe-tRNA complexes formed with either washed or reconstituted ribosomes. The effect of cations, pH, ethanol, temperature and inhibitors of peptide bond formation on [G-3H]gougerotin binding to E. coli and S. cerevisiae ribosomes has also been studied. Only those antibiotics that act on the peptidyl transferase centre of both prokaryotic and eukaryotic ribosomes (blasticidin S, sparsomycin and actinobolin) completely inhibit the binding of [G-3H]-gougerotin. In contrast, griseoviridin strongly enhances [G-3H] gougerotin binding to both types of ribosomes.
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Abstract The interaction of ribosomes with the membrane of the endoplasmic reticulum (ER) has been shown to be sensitive to puromycin and high salt1-4 as well as proteases1,5. This implies that protein-protein interactions, beyond that of the nascent chain with the translocation machinery, mediate ribosome binding. Rough microsomes, from which ribosomes have been removed (stripped), are able to rebind ribosomes, under the appropriate conditions, to pre-stripped levels5-7. To measure this rebinding, radiolabeled ribosomes are added to stripped membranes, and bound ribosomes are separated from free ribosomes by flotation of the membranes in a sucrose gradient7 In this way, the rebinding reaction can be quantified and characterized.
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