Translation Of Poly Research Articles

Effect of polyadenylic acid on the functional half-life of encephalomyocarditis virus RNA during translation

The translation of poly(A)-rich and poly(A)-poor populations of Encephalomyocarditis viral RNA was studied in Ehrlich ascites cell-free extracts. The poly(A)-rich viral RNA was translated 2–3 times more efficiently than the poly(A)-poor RNA. Both viral RNA populations were found to be similar with respect to susceptibility to nuclease attack as well as their ability to initiate and carry out protein synthesis early in the translation reaction. Later in the reaction, however, translation of the poly(A)-poor RNA was markedly reduced whereas translation of the poly(A)-rich RNA continued. These results are consistent with the hypothesis that poly(A) plays a role in the re-utilization and longevity of mRNA.

Influence of Inhibitors of Protein Synthesis on Zinc Metabolism

SummaryThe drugs actinomycin D, chloramphenicol, cordycepin, and cyclohex-imide were evaluated for their relative effectiveness in blocking zinc binding by liver and mucosa following parenteral zinc. All the drugs, except chloramphenicol, were effective. The majority of the additional zinc bound following parenteral zinc was accounted for as metallothionein (MT) zinc. The inhibition of zinc binding by the drugs used was restricted to that associated with MT. The results collectively indicate that zinc uptake in liver and intestinal cells involves nonmitochondrial transcription and translation of poly(A)-containing RNA. Some of this RNA appears to code for MT.

Decrease in levels and rates of synthesis of tubulin and actin in developing rat brain

The cytoplasmic and particulate tubulin content of postnatal rat brains was determined at various stages of development. The amount of tubulin in the soluble fraction was found to increase after birth and levels off at the age of 10–15 days, while the total protein content is still increasing. Indeed, the percentage of tubulin in the soluble fraction is about 33% at birth, stays at this value until day 10, and then decreases to 20% between days 10 and 15. On the other hand, the rate of increase in the level of the particulate tubulin parallels that of the total particulate proteins, and hence there is no change in the percentage of particulate tubulin during brain development. There was close agreement between the tubulin values obtained by the [ 3H]-colchicine binding assay and those obtained by electrophoretic resolution in sodium dodecylsulfate-polyacrylamide gels. Polyacrylamide gel electrophoresis was also utilized to determine actin levels in developing brains. The percentage of cytoplasmic brain actin also decreased with the age of the rats, from a value of 20% at birth to 10% at day 30, while the percentage of the particulate actin remained constant. The decline in the percentage of cytoplasmic tubulin and actin during brain development can be accounted for by reduction in the proportions of the respective mRNA species. Translation of poly (A)-rich brain mRNA in a wheat-germ cell-free system showed that the percentages of tubulin and actin synthesized decreased gradually with age. Similar results were obtained by analyzing the proteins produced by isolated brain polysomes in a brain cell-free system.

Translation of poly(A)-containing and poly(A)-free messenger RNA for phenylalanine ammonia-lyase, a plant-specific protein, in a reticulocyte lysate

Polyribosomal RNA from light-induced parsley cell suspension cultures was incubated in a rabbit-reticulocyte lysate. The radioactive proteins formed in vitro were treated with a specific antiserum prepared for purified phenylalanine ammonia-lyase, an enzyme which is not found in animals. Analysis of the immunoprecipitate by gel electrophoresis in the presence of dodecyl sulfate showed a single peak of radioactivity at the position of the subunits of the enzyme. No detectable amount of this protein was synthesized with RNA from parsley cells which had been kept in the dark. The polyribosomal RNA was fractionated into poly(A) + and poly(A) − RNA. Both fractions contained messenger RNA for phenylalanine ammonia-lyase, as determined with the reticulocyte lysate. The total template activity of each RNA fraction was assessed in a cell-free extract from wheat germ. About 20–40% of the total mRNA activity was associated with the poly(A) + RNA, and 60–80% with the poly(A) − RNA. Both fractions stimulated the synthesis of radioactive proteins with molecular weights ranging from 10 000 to 80 000; the size distributions of these proteins on polyacrylamide gels were very similar. The results indicate that (i) the mammalian cell-free system accepted plant mRNA for translation, (ii) considerable amounts of translatable, polyribosomal mRNA for phenylalanine ammonia-lyase were present only in irradiated parsley cells, and (iii) large portions of the enzyme-specific and the total mRNAs of the polyribosomes consisted of poly(A) − RNA.

Yeast cytochrome c messenger RNA. In vitro translation and specific immunoprecipitation of the CYC1 gene product.

An assay based upon indirect immunoprecipitation has been developed for yeast cytochrome c and apocytochrome c. The specificity of this assay was demonstrated by its ability to selectively precipitate cytochrome c from an autolysate of yeast cell proteins. Translation of the polypeptide chain of cytochrome c in a wheat germ extract programmed with yeast poly(A) RNA was demonstrated using this immunoprecipitation assay. Translation of poly(A) RNA from yeast strains carrying nonsense mutations in the cyc1 gene yielded in vitro cytochrome c polypeptides which were shorter than the wild type protein by the amount expected for polypeptide chains which had terminated at the nonsense codon. The in vivo rate of cytochrome c synthesis was shown to be 6-fold greater in derepressed cells than in glucose-repressed cells. The 6-fold difference is sufficient to account for the 6-fold higher level of cytochrome c in derepressed than in repressed cells. The level of translatable cytochrome c mRNA is at least 4 times as high in derepressed as in glucose-repressed cells, suggesting that regulation occurs at some step in the synthesis of this messenger.

Post-transcription control of isocitrate lyase induction in the eukaryotic alga Chorella fusca.

WE have shown previously1 that poly(A)-containing RNA can be isolated from the green alga Chlorella fusca 8p (ref. 2) and that this RNA has messenger-like properties, as it is polydisperse and stimulates polypeptide synthesis in vitro in a wheat embryo system. We show here that the Chlorella protein isocitrate lyase has been identified as a product of in vitro translation of poly(A)-containing RNA, so confirming messenger function in this fraction. We have used the in vitro translation system as a means of assaying for the presence of isocitrate lyase message sequences in poly(A)-containing RNA isolated from Chlorella cultured under different conditions, and we report here that slow-growing non-induced cells may contain high levels of mRNA for this enzyme although the enzyme is not synthesised.

Translation of poly(riboadenylic acid)-enriched messenger RNAs from the yeast, Saccharomyces cerevisiae, in heterologous cell-free systems.

Poly(riboadenylic acid) [poly(A)] enriched messenger RNAs from yeast have been used to direct the synthesis of yeast polypeptides in mouse Krebs II ascites and wheat embryo extracts. Both cell-free systems, synthesize polypeptides over a molecular weight range of 10,000-100,000. Autoradiograms of sodium dodecyl sulfate-polyacrylamide slab gels used to fractionate [35S]methionine-labeled in vitro products reveal that about 25 major bands (each of them possibly representing multiple polypeptides) are produced by each cell-free system. Each of these coelectrophoreses with a major polypeptide labeled in vivo or in a yeast lysate. These results suggest that cell-free translational machinery from eukaryotes is not able to discriminate in an all or none fashion against messenger RNAs which are available to it. While yeast poly(A)-enriched messenger RNA directs the synthesis polypeptides over approximately the same molecular weight range in both cell-free systems, the wheat germ system directs the incorporation of 45 times the amount of [3H]serine into Cl3CCOOH-precipitable polypeptides. This is in contrast to the 2.5-fold more efficient translation of hemoglobin mRNA in the wheat embryo extract. Thus, the extract from mammalian cells is able to translate mRNA from a lower plant with a much lower efficiency than it translates hemoglobin mRNA, and at a lower efficiency than is observed using a cell-free system from wheat embryos. This indicates that the wheat embryo system is the one of choice for translation of yeast messenger RNA.

Translation of the UGA triplet in vitro by tryptophan transfer RNA's

Tryptophan transfer RNA from the UGA-suppressing strain of Escherichia coli CAJ64 was purified and assayed for suppressor activity in vitro in two ways: by translation of the bacteriophage T4 lysozyme messenger RNA bearing a UGA mutation, and by translation of poly(U-G-A). Purified tRNA Trp, and no other fraction, stimulates lysozyme synthesis 30-fold above the level seen when comparable amounts of tryptophan tRNA from the non-suppressing strain, CA244, were added; it also translates poly(U-G-A) as polytryptophan more efficiently than the su − tRNA. Tryptophan tRNA from the non-suppressing strain is active in the assays but far less so than CAJ64 tRNA Trp, and this is consistent with the leakiness of su − strains. Since the nucleotide sequences of these tryptophan tRNA's are known (Hirsh, 1971), it is concluded that tRNA with a CCA anticodon recognizes the UGA triplet and this recognition is improved by a nucleotide change elsewhere in the molecule.

A protein factor stimulating binding and translating of polyuridylic acid by Escherichia coli ribosomes

Exposure of Escherichia coli MRE-600 ribosomes to low-ionic-strength (1 mM) Tris-acetate causes release of about 10 % of their proteins. Removal of this fraction results in a decrease in binding of poly U and in incorporation of phenylalanine by the ribosomes. Adding this fraction to the depleted ribosomes restores both activities. DEAE-cellulose chromatography reveals that this fraction contains a few acidic proteins. Only the one, which elutes at 0.175 M NaCl enhances binding and translation of poly U by the ribosomes. It also has the ability to bind poly U in the absence of ribosomes. Use of [ 3H]leucine-labeled factor shows that it interacts with the depleted 30-S ribosomal subunit. The binding activity of depleted ribosomes supplemented with the protein factor is higher than the binding activities of either the ribosomes or the factor alone. This increase suggests cooperative effect in the reconstituted system. Polyphenylalanine synthesis shows that the binding of poly U took place at the mRNA site on the supplemented ribosomes.

Functional and Structural Studies of Membrane-bound and Free Ribosomes from Rat Spleen

Abstract The functional and structural properties of sodium deoxycholate-liberated bound and free ribosomes from rat spleen have been compared in vitro. The endogenous amino acid-incorporating activity of bound ribosomes relative to that of free ribosomes (B:F) varies only slightly (71 to 103%) under a variety of isolation and incubation conditions. However, the net exogenous activity for 14C-phenylalanine with the use of polyuridylic acid (poly U) as messenger varies widely (B:F, 11 to 75%) and is greatly dependent on experimental conditions. Minimal translation of poly U and the lowest B:F ratio are seen when the source of supernatant factors is a spleen pH 5 fraction. Maximal translation of poly U and the highest B:F ratio are achieved with a liver pH 5 fraction, isolation of ribosomes in the presence of a rat liver supernatant, the omission of centrifugation through 2 m sucrose, and the addition of a 0.5 m NH4Cl wash. Relative to the minimal system, incorporation is enhanced 54-fold for bound ribosomes and 8-fold for free ribosomes when these optimal conditions are used. Rat spleen bound and free ribosomes are immunologically identical, have similar sedimentation coefficients, similar contents of RNA and nucleotide bases, and virtually identical patterns of polyribosomes and structural and rapidly labeled RNAs. These results caution that different ribosome populations may seem more or less alike functionally, depending upon the particular conditions of isolation and incubation.