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PubMed1982

Studies on the catalytic properties of the calcium-dependent endoribonuclease of Trypanosoma brucei cytoplasm.

A calcium-dependent endoribonuclease isolated from Trypanosoma brucei cytoplasm has been further characterized. The products of exhaustive endonuclease digestion of poly(A), which the enzyme cleaved preferentially, were small oligonucleotides terminated by a 5'-phosphoryl group. Its apparent Km value was 0.8 mM using poly (A), and 3.3 mM using transfer RNA. The base specificity of the endoribonuclease was further verified by using synthetic heteropolyribonucleotides as substrates. The enzymatic reaction was inhibited by polyamines, polyanions, iodoacetic acid and the heavy metals, but only very slightly by N-ethylmaleimide and iodoacetamide. A possible implication of the enzyme in the turnover of messenger RNA is discussed.

Animals↗
PubMed1982

Hydrogen exchange kinetics of nucleic acids. Double and triple helices with Hoogsteen-type basepairs.

The kinetics of hydrogen-tritium exchange reaction have been followed by a Sephadex technique of a double-helical poly(ribo-2-methylthio-adenylic acid) . poly(ribouridylic acid) complex with the Hoogsteen-type basepair. Only one hydrogen in every 2-methylthio-adenine . uracil basepair has been found to exchange at a measurably slow rate, 0.023 s-1 (at 0 degrees C), which is, however, much greater than that for a double-helix with the Watson-Crick type A . U pair. The kinetics of hydrogen-tritium exchange were also examined by triple-helical poly(rU) . poly(rA) . poly(rU) which involves both the Watson-Crick and Hoogsteen basepairings. Here, three hydrogens in every U . A. U base triplet have been found to exchange at a relatively slow rate, 0.0116 s-1 (at 0 degrees C). The kinetics of hydrogen-deuterium exchange reactions of these polynucleotide helices have also been followed by a stopped-flow ultraviolet absorption spectrophotometry at various temperatures. On the basis of these experimental results, the mechanism of the hydrogen exchange reactions in these helical polynucleotides was discussed. In the triple helix, the rate-determining process of the slow exchange of the three (one uracil-imide and two adenine-amino) hydrogens is considered to be the opening of the Watson-Crick part of the U. A. U triplet. This opening is considered to take place only after the opening of the Hoogsteen part of the triplet.

Base Composition↗
PubMed1986

Formylmethionyl-tRNA binding to 30 S ribosomes programmed with homopolynucleotides and the effect of translational initiation factor 3.

Binding of the polynucleotides poly(U), poly(X) and poly(dT) to 30 S ribosomes of Escherichia coli triggers IF2-dependent binding of initiator-tRNA (fMet-tRNA) to these particles. Poly(A) and poly(C) are inactive. A minimum chain-length of approximately 100 residues in poly(U) is required for full activity in fMet-tRNA binding, although much shorter polymers bind tightly to 30 S particles and do stimulate the binding of acPhe-tRNA. The stimulation of fMet-tRNA binding to 30 S ribosomes is strongly reduced under conditions where the polynucleotides adopt secondary structure. Complexes containing fMet-tRNA and the non-cognate codon UUU or XXX are destabilized by IF3, whereas the formation of such a complex containing an AUG codon is slightly enhanced by the factor. Consistent with previous observations, it was found that all model initiation complexes containing acPhe-tRNA are strongly destabilized by IF3, even when the cognate codon (UUU) is present. Our results suggest that IF3 counteracts 'unnatural' initiation events in vitro and suggest a regulatory role for this factor in vivo.

Anticodon↗
PubMed1992

An inhibitor of elongation factor G (EF-G) GTPase present in the ribosome wash of Escherichia coli: a complex of initiation factors IF1 and IF3?

An inhibitor of elongation factor G (EF-G) GTPase isolated from the ribosome wash of Escherichia coli was shown to stimulate the poly(A,U,G)- and initiation factor 2 (IF2)-dependent binding of N-formyl-[35S]Met-tRNAfMet to ribosomes. In the presence of saturating amounts of the EF-G GTPase inhibitor, neither addition of initiation factor 1 (IF1) nor addition of initiation factor 3 (IF3) caused a further stimulation of the formation of N-formyl-[35S]Met-tRNAfMET/poly(A,U,G)/ribosome complexes. Both IF1 and IF3 were shown to inhibit ribosome-dependent EF-G GTPase, especially when both initiation factors were added either in absence or in the presence of initiation factor 2 (IF2), poly(A,U,G) and N-formyl-Met-tRNAfMet. Therefore, we conclude that the EF-G GTPase inhibitor consisting of two polypeptide subunits with apparent molecular masses of 23,000 and 10,000 Da is a complex of initiation factors IF1 and IF3. The inhibition of EF-G GTPAse by IF3, but not the effects of IF1 in the presence or absence of IF3 could be reversed by increasing the Mg(2+)-concentration as already shown for the EF-G GTPase inhibitor. Therefore, IF1 as well as the EF-G GTPase inhibitor do not influence the ribosome-dependent EF-G GTPase by affecting the association of ribosomal subunits.

Bacterial Proteins↗
PubMed1993

Structural arrangement of the codon-anticodon interaction area in human placenta ribosomes. Affinity labelling of the 40S subunits by derivatives of oligoribonucleotides containing the AUG codon.

Using the derivatives of the oligoribonucleotides pAUGUn and AUGUnC (n = 0; 3) bearing an alkylating group at either the 5' or 3' end, respectively (mRNA analogues), the affinity labelling of the human placenta 40S ribosomal subunits has been investigated in model initiation complexes obtained in the presence of the ternary complex eIF-2.GTP.Met-tRNA(fMet). The regions of 18S rRNA and ribosomal proteins labelled with these mRNA analogues were identified. The sites of covalent attachment of the pAUGUn derivatives with a reactive group at the 5' end were located between 18S rRNA positions 976 and 1164. The derivative of AUGU3C with an alkylating group at the 3' end modified 18S rRNA mainly at the 593-673 region. The main targets of the 3' end derivative of AUGC were located between positions 1610 and 1869. The proteins S3/S3a, S6, S7 and S14/S15 were modified by both types of the oligoribonucleotide derivatives regardless of the point of the reactive group attachment to the oligonucleotide moiety. The proteins S2 and S4 were modified by both the 3' end derivative of AUGC and 5' end derivative of pAUGU3; and the protein S8 was modified by the 3' end derivative of AUGC. The proteins S5 and S9 were labelled by the 5' end derivative of pAUGU3, and the protein S17 was modified by the 5' end derivative of pAUG.

Affinity Labels↗
PubMedDate not supplied

VIP induces in HT-29 cells 2'5'oligoadenylate synthetase and antiviral state via interferon beta/alpha synthesis.

Vasoactive intestinal peptide (VIP), composed of 28 amino acids, is a multifunctional neurotransmitter. We have demonstrated here that its action on human transformed colonic epithelial (HT-29) cells is mediated through the induction of interferon (IFN) synthesis. We have found that these cells have a functional receptor for IFN alpha 2; binding was specific to either IFN alpha 2 or IFN beta but not to IFN gamma. VIP induced the 2'5'oligoadenylate synthetase (2'5'A synthetase) and the antiviral state with the same efficiency as poly (I).poly (C). The induction of 2'5'A synthetase activity required cellular RNA and protein synthesis, and the maximum induction occurred with 10(-7) M VIP at 24 h. VIP, like some IFN inducers, induced the synthesis of the 70 hsp which, however, preceded the expression of 2'5'A synthetase. VIP treatment caused the induction and secretion of IFN, having a titer value of 32 international units/ml. This IFN has been identified as type beta/alpha, because both 2'5'A synthetase and the antiviral activities were abolished by anti-human IFN beta/alpha antibodies, but not by anti-IFN gamma antibodies. Thus the pathway of VIP action on HT-29 cells may be outlined as 1) binding of VIP, 2) synthesis of 70 hsp, 3) induction of IFN synthesis and its secretion, 4) binding of the secreted IFN to cell surface receptors and 5) turning on the induction of 2'5'A synthetase and antiviral activities.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

The origin and early evolution of nucleic acid polymerases.

The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA polymerase beta' subunit and its homologues is discussed. We show that in the DNA-dependent RNA polymerases from the three cellular lineages a very conserved sequence of eight amino acids also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein is present. The optimal conditions for the replicase activity of the avian myeloblastosis virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is also discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Enzymatic polymerization of 5-mercuriuridine-5'-diphosphate with polynucleotide phosphorylase from E. coli.

We report the polymerization of 5-mercuriuridine-5'-diphosphate (ppUHgX), in the presence of an excess of beta-mercaptoethanol, with polynucleotide phosphorylase from E. coli. A degradation of mercurated nucleotides with mercaptans was observed and about 30% of incorporation of mercury in the polymer was obtained after treatment with pancreatic RNase. The influence of ppUHgX and pUHgX with or without beta-mercaptoethanol was also studied on the polymerization of UDP. The ppUHgX did not polymerize in the absence of beta-mercaptoethanol.

Chromatography, Thin Layer↗
PubMed1990

Different specificities of ribonuclease II and polynucleotide phosphorylase in 3'mRNA decay.

We review recent evidence on the in vivo and in vitro mRNA degradation properties of 2 3'-exonucleases, ribonuclease II and polynucleotide phosphorylase. Although secondary structures in the RNA can act as protective barriers against 3' exonucleolytic degradation, it appears that this effect depends on the stability of these structures. The fact that RNase II is more sensitive to RNA secondary structure than PNPase, could account for some differences observed in messenger degradation by the 2 enzymes in vivo. Terminator stem-loop structures are often very stable and 3' exonucleolytic degradation proceeds only after they have been eliminated by an endonucleolytic cleavage. Other secondary structures preceding terminator stem-loop seem to contribute to mRNA stability against exonucleolytic decay.

Base Sequence↗
PubMed1990

Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12.

Polynucleotide phosphorylase (PNPase) has been studied in detail since its discovery in 1955 [1]. In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coli, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene. In vitro phosphorolysis, polymerization and exchange activities of the partially purified Pnp-200 enzyme are all reduced to 30-40% of wild-type activity at 50 degrees C compared to 32 degrees C. The pnp-200 mutation alone does not affect cell growth or mRNA stability. A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism (rnb-500 and ams-1) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature.

Escherichia coli↗
PubMed1991

Different specificities of ribonuclease II and polynucleotide phosphorylase in 3'mRNA decay.

We review recent evidence on the in vivo and in vitro mRNA degradation properties of 2 3'-exonucleases, ribonuclease II and polynucleotide phosphorylase. Although secondary structures in the RNA can act as protective barriers against 3' exonucleolytic degradation, it appears that this effect depends on the stability of these structures. The fact that RNase II is more sensitive to RNA secondary structure than PNPase, could account for some differences observed in messenger degradation by the 2 enzymes in vivo. Terminator stem-loop structures are often very stable and 3' exonucleolytic degradation proceeds only after they have been eliminated by an endonucleolytic cleavage. Other secondary structures preceding terminator stem-loop seem to contribute to mRNA stability against exonucleolytic decay.

Base Sequence↗
PubMed1976

A calorimetric study of polyguanylic acid at neutral pH.

The structure of polyguanylic acid (poly G) at neutral pH has been studied by optical and calorimetrical methods. It can be shown that diverging from earlier findings Poly G reversibly undergoes a cooperative thermal transition. Thermal denaturation curves are recorded at 253 nm as a function of the sodium ion concentration. The denaturation enthalpy of poly G in dilute aqueous solution is determined to 2.2 kcal/mole g. It is concluded, that the part of the ordered poly G structure, which gives rise to a temperature dependent cooperative transition, arises from stacking interactions of adjacent bases in the single strand.

Calorimetry↗
PubMed1985

Raman scattering from nucleic acids adsorbed at a silver electrode.

Adsorption of nucleic acids at a silver electrode polarized to -0.6 to -0.1 V (vs. Ag/AgCl) was investigated by means of surface enhanced Raman scattering (SERS) spectroscopy. Single-stranded polyriboadenylic acid and thermally denaturated DNA adsorbed at the silver electrode yield two intense bands at 734 and 1335 cm-1 on the SERS spectra. These bands, assigned to the vibrations of adenine residue rings, were much less intense if the SERS spectra were recorded for double-helical complex polyadenylic X polyuridylic acid and native DNA. Moreover, the courses of alkaline denaturation of DNA and its digestion by deoxyribonuclease I were observed by SERS spectroscopy. The results were interpreted as support for the view that intact double-helical segments of nucleic acids are not denatured or destabilized due to their adsorption at the positively charged and roughened surface.

Animals↗
PubMed1986

Chemically reversible electroreduction of guanine in a polynucleotide chain.

It was shown that synthetic polynucleotides containing guanine display in cyclic voltammetry (CV) an anodic peak close to -0.3 V (against a saturated calomel electrode). A condition for the appearance of this peak is the previous polarization of the mercury electrode to sufficiently negative potentials (around -1.8 V). The results of CV measurements with electrode polarization by repeated cycles indicate that in negative potentials there is a reduction of guanine residues and in the anodic process reoxidation of the reduction product to guanine. This chemically reversible process takes place even when a polynucleotide contains adenine and/or cytosine residues in addition to guanine, where reduction leads to the formation of products blocking the electrode surface.

DNA↗
PubMed1987

The binding of Mg(II) and Ni(II) to synthetic polynucleotides.

Equilibria and kinetics of the interactions of Mg2+ and Ni2+ with poly(U), poly(C) and poly(I) have been investigated at 25 degrees C, an ionic strength of 0.1 M, and pH 7.0 or 6.0. Analogous studies involving poly(A) were reported earlier. All binding equilibria were studied by means of the (usually small) absorbance changes in the ultraviolet range. This technique yields apparent binding constants which are fairly large for the interaction of Ni2+ with poly(A) (K = 0.9 X 10(4) M-1) and poly(I) (K approximately equal to 2 X 10(4) M-1) but considerably lower for the corresponding Mg2+ systems, Mg2+-poly(A) (K = 2 X 10(3) M-1) and Mg2+-poly(I) (K = 280 M-1). Each of the two pyrimidine nucleotides binds both metal ions with about the same strength (K approximately equal to 65 M-1 for poly(U) and K near 600 M-1 for poly(C]. In the case of poly(C) the spectral changes deviate from those expected for a simple binding equilibrium. In addition, the binding of Ni2+ to the four polynucleotides was measured by using murexide as an indicator of the concentration of free Ni2+. The results obtained by this technique agree or are at least consistent with those derived from the ultraviolet spectra. Complications are encountered in the binding studies involving poly(I), particularly at higher metal ion concentrations, obviously due to the formation of aggregated poly(I) species. Kinetic studies of the binding processes were carried out by the temperature-jump relaxation technique. Measurable relaxation effects of time constants greater than 5 microseconds were observed only in the systems Ni2+-poly(A) and Ni2+-poly(I). Such not-too-fast reaction effects are expected for processes which include inner-sphere substitution steps at Mg2+ or Ni2+. The relaxation process in Ni2+-poly(I) is characterized by (at least) four time constants. Obviously, the complicated kinetics again include reactions of aggregated poly(I). The absence of detectable relaxation effects in all other systems (except Mg2+-poly(I), the kinetics of which was not investigated) indicates that inner-sphere coordination of the metal ions to specific sites of the polynucleotides (site binding) does not occur to a significant extent. Rather, the metal ions are bound in these systems mainly by electrostatic forces, forming a mobile cloud. The differences in binding strength which are nevertheless observed are attributed to differences in the conformation of the polynucleotides which result in different charge densities.

Kinetics↗
PubMed1988

Kinetic and thermodynamic investigation of the (dien)Pd(II)-polycytidylic acid system.

The reaction between (dien)PdCl+ and polycytidylic acid was studied using spectroscopic and stopped-flow methods. In neutral solution, the palladium complex binds at the N3 site of the cytosine base and causes a noncooperative disruption of the ordered helical structure of poly(C). Interaction at the phosphate group of the polynucleotide was also demonstrated by using the dye acridine orange as an indicator. The results of this study show that the mechanism previously proposed for cytidine and CMP can be applied to poly(C), taking into account particular features of the polymer (polyelectrolytic nature, structure, etc.). In particular, electrostatic effects seem to play a major role in the interaction with metal ion complexes like (dien)Pd(II).

Acridine Orange↗
PubMedDate not supplied

Template activity of poly(2'-fluoro-2'-deoxyinosinic acid) for murine leukemia virus reverse transcriptase.

The 2'-substituted analog of poly(inosinic acid) ((I)n), poly(2'-fluoro-2'-deoxyinosinic acid) ((dIfl)n) served as an effective template for the RNA-directed DNA polymerase (reverse transcriptase) from Moloney murine leukemia virus. When assayed under the same conditions, the parent compound (I)n showed little, if any, template activity. In the presence of other templates, i.e. poly (2-O-methylcytidylic acid), (dIfl)n could also assume the role of primer for the reverse transcriptase reaction, whereas, again, (I)n failed to do so.

Dose-Response Relationship, Drug↗