Fractionation of the complementary strands of coliphage T4 DNA based on the asymmetric distribution of the poly U and poly U,G binding sites.
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Centrifugation, Density Gradient↗
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Effect of E. coli ribosomal protein S1 on the fidelity of the translational elongation step: reading and misreading of poly(U) and poly(dT).
Ribosomal protein S1 was selectively removed from E. coli ribosomes by affinity chromatography and the effect of added S1 on the translation of poly(dT) [which is read as poly(U) in the presence of neomycin] and on the misreading of poly(U) and poly(dT) were examined. S1 enhances the translation of poly(dT) at low template concentration, which is similar to the effect of S1 on poly(U) translation. The misreading of poly(dT) by E. coli ribosomes is at a lower level than is the case with poly(U). This low misreading is the same for "S1-dependent" and "S1-independent" modes of translation. On the other hand, the misreading of poly(U) is significantly reduced when S1 is present. These results thus indicate that S1 not only facilitates the binding of mRNA to the ribosome as already known, but also plays a role in the correct codon-dependent selection of aminoacyl-tRNA.
Bacterial Proteins↗
[Study of fusion of bacteriophage f2 double-stranded RNA, poly(A).poly(U), and poly(G).poly(C) in the presence of tetraethylammonium bromide].
The data on the dependence of the melting curve parameters of double-stranded RNA (replicative form of RNA of f2 bacteriophage) poly(A) times poly(U) and poly(G) times poly(C) on the concentration of (C2H5)4NBr were obtained. The RNA melting range width is shown to pass through the minimum value T =2.1+/-0.1degrees at the point of inversion of relative stability of GC and AU pairs that corresponds to 4.0+/-0.1 M concentration of (C2H5)4NBr. Using the melting temperatures of poly(A) times poly(U) and poly(G) times poly(C) the rependence of Tgc-Tau parameter on (C2H5)4NBr concentration was shown. It was concluded from these data that the effect of the double-stranded RNA stacking heterogeneity was negligible in the 0-3 M range of (C2H5)4NBr concentration. Melting curves of RNA were obtained at various values of Tgc-Tau parameter. It was shown that the profile of fine structure of melting curves depends on the value of Tgc-Tau parameter.
Bacteriophages↗
Proton magnetic resonance studies of the binding of oligopeptides containing tryptophan to polyribonucleotides poly A, poly U and poly C.
The binding of oligopeptides Lys-Trp-Gly-Lys OtBu, Lys-Gly-Trp-Lys OtBu and Lys-Trp-Lys to Polyadenylic, Polycytidylic and Polyuridylic acid has been studied by Proton NMR at 90 MHz and 500 MHz at oligopeptide/Polynucleotide ratios ranging from 0.01 to 0.20 at 275-365 K. Downfield shift of 0.01-0.2 ppm at 296 K of the H2, H8 and H1' resonances of Poly A due to binding with oligopeptides is accompanied by a marked narrowing of resonance lines of Poly A. The ring protons of tryptophan shift upfield by 0.3-0.6 ppm at 296 K on binding to Poly A. Changes in chemical shift of both adenine and tryptophan protons on binding are much smaller at 355 K than that at 275 K. These observations are ascribed to intercalation of the tryptophan ring in the adenine bases resulting in partial destacking of adenine bases in Poly A. Using the magnetic anisotropy ring current shifts, an overlap geometry of tryptophan ring in the adenine has been proposed. Addition of oligopeptides to Poly C and Poly U, on the other hand, suggests that tryptophan ring does not stack in Poly U and Poly C.
Magnetic Resonance Spectroscopy↗
Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol.
Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+. Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).
Binding Sites↗
Oligo(U)- and poly(U)-containing RNA of wheat embryo.
A UMP-rich RNA fraction was separated from the bulk cellular RNA by affinity chromatography of wheat embryo total RNA. The obtained preparation was heterogeneous and contained polyribonucleotide chain segments which were resistant to RNAase T1 and consisted mainly of UMP residues (87 mol%). The UMP-rich segments were of various sizes, including large oligonucleotides and polynucleotides (up to approx. 150 nucleotides in length). The oligo(U)- and poly(U)-containing RNA fraction occurred in a low amount (approx. 1% of total RNA) both in dry and germinating wheat embryos. However, at the onset of germination, labelled precursors were preferentially incorporated into the UMP-rich RNA species. The early-synthesized RNA appeared and underwent a considerable degradation within the cell nuclei. It is assumed that both delayed maturation of structural gene transcripts and rapid transcription of regulatory gene units during initial germination stages contribute to the transient abundance of newly made UMP-rich RNA in the early wheat embryos.
Base Sequence↗
Purification and characterization of a ribonuclease specific for poly(U) and poly(C) from the larvae of Ceratitis capitata.
A specific ribonuclease was detected and purified to homogeneity from six-day-old larvae of the insect Ceratitis capitata and its homogeneity was checked by analysis in polyacrylamide gels in the presence of sodium dodecyl sulfate. The nuclease specifically degrades poly(U) and poly(C) whilst it fails to do so with other single-stranded homopolyribonucleotides. The enzyme has a pH optimum in the region 7-9 and relative molecular mass of about 25,000. The effect of this ribonuclease on the integrity of RNAs isolated from six-day-old larvae or rat liver was also studied.
Animals↗
Activation of the murine monocyte/macrophage cell line, J774A1 by poly(A).poly(U): I. Binding of poly(A).poly(U) and induction of oligo-2',5'-adenylate synthetase.
Binding and internalization of the synthetic double-stranded complex poly(A).poly(U) were studied on a murine monocyte/macrophage cell line J774A1. Poly(A).poly(U) increased in a dose-dependent fashion the oligo-2',5'-adenylate synthetase demonstrating that those cells were responsive to this agonist. Binding of [32P]poly(A).[32P]poly(U) to the cells reached an apparent kinetic equilibrium within 4 h and was saturable (apparent Kd = 9.99 +/- 0.09.10(-2) g/l and Bmax 13.3 +/- 5.3.10(-3) g/l per 10(6) cells) and temperature-dependent. The binding of poly(A).poly(U) was competitively inhibited by various polynucleotides but not by other structurally unrelated compounds. Analysis of cell-associated [32P]poly(A).[32P]poly(U) demonstrated a minimal degradation of this polyribonucleotide over a 4-h incubation period. Autoradiography of cells incubated with [3H]poly(A).[3H]poly(U) revealed that poly(A).poly(U) was internalized and migrated to cell nuclei. These results suggest that poly(A).poly(U) is internalized in J774A1 cells via an endocytotic process.
2',5'-Oligoadenylate Synthetase↗
Identification of the amide II band in the cis-amide 1-methyluracil, in poly(U), and in poly(A)-poly(U).
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Amides↗
Interaction of poly-L-lysine with nucleic acids. II. Poly(A+U), poly(A+2U), and rice dwarf virus RNA.
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Chemical Phenomena↗
Stimulation of B cells by poly A: poly U and poly I: poly C in vitro.
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Animals↗
Proteins of small subunits of rat liver ribosomes that interact with poly(U). II. Cross-links between poly(U) and ribosomal proteins in 40 S subunits induced by UV irradiation.
(1) When rat liver 40 S ribosomal proteins in 6 M urea were were mixed with poly(U) at an appropriate ratio, a precipitate was formed which was also insoluble in the sample solution for two-dimensional acrylamide gel electrophoresis. Analyses by two-dimensional acrylamide gel electrophoresis showed that S7 and S10 proteins (according to our numbering system) had disappeared selectively from the fraction soluble in 6 M urea. These two proteins were present in the fraction insoluble in 6 M urea, and became soluble in the sample solution after treating it with RNase. The results suggest that S7 and S10 proteins have strong affinities for poly(U). When rat liver 40 S subunits were incubated with poly(U), similar results were obtained. (2) After incubation of 40 S subunits with [3H]poly(U) and then with unlabeled poly(U), UV irradiation cross-linked poly(U) to the protein moiety of the 40 S subunit. When the protein fraction insoluble in the sample solution for two-dimensional electrophoresis was prepared from 40 S subunits cross-linked to poly(U) and then subjected to two-dimensional acrylamide gel electrophoresis after RNase treatment, S7 and S10 proteins were detected on the gel. In addition to the S7 protein spot, a triangular area spreading from the spot to the origin contained radioactivity. The results suggest that poly(U) is cross-linked to S7 protein and oligo(U) fragments bound to S7 protein affect its electrophoretic mobility. (3) Ribosomal proteins were prepared from 40 S subunits cross-linked to carrier-free [3H]poly(U) and analyzed by three-dimensional acrylamide gel electrophoresis (Terao, K. & Ogata, K. (1975) Biochim. Biophys. Acta 402, 214--229) after RNase treatment. It was found that S7, S6, and S15 proteins are cross-linked to poly(U). From the results of the present and preceding experiments it is concluded that S7 is the poly(U)-binding protein. The possibility that other proteins in 40 S ribosomal subunits interact with poly(U) is discussed.
Animals↗
Correlation between poly(U) misreading and poly(dT) translation efficiency in E coli cell-free systems.
A positive correlation between poly(U) misreading and efficiency of poly(dT) translation has been revealed in cell-free systems from wild-type E coli and streptomycin--resistant mutants with altered ribosomal protein S12. Different factors promoting misreading of poly(U) such as aminoglycoside antibiotics and Mg2+ ions also stimulate poly(dT) translation. The effect of the antibiotics on poly(U) translation efficiency and misreading as well as on poly(dT) decoding is characterised by the same order: neomycin greater than kanamycin greater than streptomycin. S12 mutants ribosomes are less erroneous in poly(U) translation and less efficient in poly(dT) decoding. The data obtained are in good agreement with the hypothesis of stereospecific stabilization of codon-anticodon complexes by the ribosome decoding centre.
Anti-Bacterial Agents↗
The separation of three antibody populations from anti-poly(A).poly(U) antibodies elicited in mice or rabbits and antigenic features of poly(A).poly(U)).
Anti-poly(A).poly(U) antibodies in ascitic fluid of DDY mice immunized with poly(A).poly(U)-methylated bovine serum albumin complexes were fractionated into three major antibody populations, Ab-1, Ab-2 and Ab-3, by precipitating with poly(I).poly(C), poly(A).poly(U), and poly(A).2 poly(U), respectively. Antibody population one, Ab-2, reacted with various double-stranded RNAs [poly(I).poly(C), poly(A).poly(U), and rice dwarf virus ribonucleic acid (RDV-RNA)] and poly(A).2 poly(U). Ab-2 reacted with poly(A).poly(U) and poly(A).2 poly(U). Although both Ab-1 and Ab-2 reacted with poly(A).poly(U), the two populations were distinguishable by their different reactivities against chemically modified antigens and oligonucleotides. In contrast to Ab-2, acetylation at the furanose 2'-position of poly(U) resulted in a dramatic decrease in the complement fixation reactivity of Ab-2. Also, Ab-2 was capable of binding with complexes of hexa- to heptaadenylates and poly(U), whereas Ab-1 required oligoadenylates of longer chain lengths (9-10 chain length) for binding. Therefore, it appears that poly(A).poly(U) possesses unique antigenic determinants which are recognizable only by Ab-2, in addition to those determinants which are common to a variety of double-stranded RNAs.
Animals↗
Raman studies of nucleic acids. VII. Poly A-poly U and poly G-poly C.
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Base Sequence↗
The low temperature reaction of poly U and poly A.
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Adenine Nucleotides↗
Detection of polyadenylic acid sequences in viral and eukaryotic RNA(polu(U)-cellulose columns-poly(U) filters-fiberglass-HeLa cells-bacteriophage T4).
A rapid and specific technique to detect polyriboadenylic acid sequences in RNA is described. The method depends upon the ability of RNAs that contain poly(A) sequences to associate specifically with poly(U) that has been immobilized on fiberglass filters by ultraviolet irradiation. A high proportion of the transcripts synthesized in vivo and in vitro from the vaccinia virus genome contain poly(A) sequences and bind to the poly(U) filters. Similarly, DNA-like RNA from the nucleus and from the cytoplasmic polyribosomes of HeLa cells is rich in species that bind to poly(U) filters. Poly(U) immobilized on cellulose powder is useful to make columns with a high capacity for the binding and purification of poly(A)-containing RNAs.
Adenine Nucleotides↗
Release of K+ and H+ from poly U in aqueous solution upon gamma and electron irradiation. Rate of strand break formation in poly U.
Conductivity changes were found which followed the reaction of radiolytically generated OH radicals with the potassium salt of polyuridylic acid (poly U) in aqueous solution. After 60Co-gamma-irradiation the observed increase of conductivity at pH = 6.8 was shown to consist of the liberation of K+ ions from the stock of K+ ions electrostatically bound to the polyanion. The initial G(K+) is 36 and hence 6 times higher than the G value of OH radicals in N2O saturated solutions. At a poly U concentration of 60 mg l-1 half of the ion release occurred at 12 J kg-1 and nearly all ions are released at 40 J kg-1. The liberation of K+ is explained to be a consequence of the formation of chain breaks leading to an increase of the degree of dissociation. The rate of the ion release was studied under pulse radiolysis conditions. Because of the high G-value of counterion liberation and the use of conductivity as analytical quantity the method is very sensitive. With 6 mg l-1 poly U the rate could be measured even at a dose per pulse of 0.25 J kg-1. The kinetics of the ion release can be described in terms of two parallel first order reactions of comparable contribution with an average rate constant of 0.8 s-1 at 20 degrees C, 60 mg l-1 poly U and pH = 6.8 with a small contribution of slower components. In more acidic solutions, besides K+ ions H+ are also liberated since at low pH values bound K+ is replaced by H+. The rate of the ion release was found to increase with increasing replacement of K+ by H+ (kobs = 100 s-1 at pH = 3.4, 60 mg l-1 poly U and T = 18 degrees C). With potassium ion free polyuridylic acid the corresponding rate constant amounted to 220 s-1, nearly independent of pH. From the temperature dependence activation parameters for the ion release were derived (Ea = 57 kJ mol-1, A = 1.0 X 10(10) s-1 at pH = 6.8). Addition of p-benzoquinone at pH = 3.7 and dithiothreitol (DTT) at pH 6.8 were found to decrease the size of the conductivity changes and to increase the rate. The results show that p-benzoquinone and DTT react with poly U radicals and that these reactions prevent chain breaks and ion liberation. It is concluded that the rate determining step of the conductivity increase is the formation of strand breaks by a cleavage of poly U radicals and that this reaction is pH dependent. The pH dependence and the observed value for the activation energy was found to be in agreement with the behaviour of a model system for the earlier postulated C-4' mechanism for strand break formation of polynucleotides and DNA.
DNA↗