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PubMed1993

Mammalian heterogeneous ribonucleoprotein A1 and its constituent domains. Nucleic acid interaction, structural stability and self-association.

With a view toward further understanding the structure-function relationships of the eukaryotic heterogeneous ribonucleoprotein (hnRNP) A1, and in particular its multiplicity of nucleic acid-interactive domains, we have studied the nucleic acid binding properties of the globular N-domain (UP1) and sequence-repetitive, flexible C-domain, the thermal denaturation of UP1 and the concomitant effects of binding polynucleotide, and the self-associative properties of the full-length protein. Utilizing protein tryptophan fluorescence as a probe, polynucleotide binding was shown to stabilize UP1 against thermal unfolding. The denaturation profile of UP1-poly(thymidylic acid) complexes was biphasic, suggesting that unfolding of the two subdomains of UP1 can occur independently. This is in agreement with a previously proposed structure in which only one of the two UP1 subdomains binds the nucleic acid. The subdomains of UP1 can be prepared by controlled proteolysis of A1, further indicating that these two globular segments within A1 are connected by an exposed, flexible linkage. Circular dichroism measurements on UP1 confirm previous data that this portion of A1 binds single-stranded nucleic acids non-co-operatively. UP1 clearly shows a preference for single-stranded nucleic acids with a 2'-OH, since its affinity for poly(U) is three times higher than for poly(dU), and five times higher than its affinity for poly(2'-OCH3U). The nucleic acid-interactive properties of the C-domain were further examined by preparing a synthetic peptide polymer (M(r) approximately 12,000) containing about seven repeats of a 16-residue sequence, GNFGGGRGGNYGGSRG, which in turn comprises two copies of the C-terminal consensus, GN(F/Y)GG(G/S)RG. The polymer of this sequence exhibited significant affinity for the fluorescent polyribonucleotide, poly(ethenoadenylic acid), binding stoichiometrically at < or = 0.2 M-Na+. Complex formation was accompanied by an increase in aggregate formation, as indicated by the appearance of scattering. For purposes of comparison, the data were analyzed via the linear co-operative model of McGhee and von Hippel, though this model may not be fully descriptive of the protein-nucleic acid complex(es) formed in this case. In contrast to the non-co-operative binding mode of the UP1 domain, the C-polymer exhibited moderate co-operativity, comparable to that seen with full-length A1. Although addition of sufficient NaCl reversed the interaction, a sigmoidal binding isotherm could still be observed (with sufficient added polymer) at 0.8 M-NaCl. This suggests that non-electrostatic interactions contribute significantly to the free energy of binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

The refined three-dimensional structure of an insect virus at 2.8 A resolution.

The structure of the black beetle nodavirus has been refined at 2.8 A resolution by alternate use of restrained least-squares atomic coordinate refinement and phase refinement by real space averaging with the 5-fold non-crystallographic symmetry in the crystal. The coordinates were also refined by simulated annealing. The final R-factor for all data with I/sigma(I) > 4 was 22.1%. A total of 7692 atoms were refined in one icosahedral asymmetric unit which included 273 oxygen atoms of ordered water molecules. Three identical gene products of 407 amino acids form one icosahedral asymmetric unit. Each is located in a structurally unique position, identified as A, B or C, consistent with a T = 3 quasi equivalent lattice. Icosahedral pentamers are formed by A subunits while B and C subunits alternate about icosahedral 3-fold axes to form quasi hexamers. Five calcium ions are located within the icosahedral asymmetric unit and stabilize the quasi 3-fold related intersubunit contacts between A, B and C. The final model consists of coordinates for residues 56 to 379 of all three subunits and residues 20 to 31 from the C subunit only. Atom positions for the sugar-phosphate backbone were modeled for ten nucleotides close to the icosahedral 2-fold axes. Symmetry equivalent polyribonucleotides form a helical duplex at each icosahedral 2-fold axis. The three subunits display an eight-stranded beta-barrel fold, very similar to the subunit structures observed in most other icosahedral RNA viruses analyzed. Quasi equivalence is regulated by the ordered RNA and residues 20 to 31 in the C subunit to form a "flat inter subunit contact" at icosahedral 2-fold joints. The RNA and polypeptide are disordered at the quasi 2-fold joints and this results in a "bent inter subunit contact". Although similar quaternary structures were seen in T = 3 plant viruses studied, RNA did not play a role as a molecular switch in those structures. The autocatalytic, post assembly, cleavage of the initial gene product at residue Asn363/Ala364 to form a stable and infectious particle is probably the result of an acid catalyzed main-chain hydrolysis in which Asp75 is the proton donor. The reaction is initiated by assembly which places Asp75 in a hydrophobic environment created by quaternary interactions which raises its pK to 5.6. The region in which the reaction occurs is formed by an internal helical bundle that has not been seen in other virus structures.

Amino Acid Sequence↗
PubMed1995

Increase of satellite tobacco ringspot virus RNA initiated by inoculating circular RNA.

A small satellite RNA of tobacco ringspot virus (sTRSV RNA) generates circular and linear molecules of unit length and repetitive sequence, linear multimers during replication. The phosphodiester junction joining the unit satellite RNA sequences in multimeric and circular RNA resisted base-catalyzed cleavage in circles but not in linear dimers. We postulate that junctions of multimeric satellite RNA form during synthesis of the polyribonucleotide chain, whereas those of circular RNA result from a ligation reaction that introduces a group blocking the junction 2'-hydroxyl. To test the relative effectiveness of linear and circular satellite RNAs in initiating replication, we inoculated onto bean (Phaseolus vulgaris cv Black Valentine) the four possible pairs of satellite RNA molecules, one member of each pair having the wild-type sTRSV RNA sequence and the other that of the replicating mutant 51AG/212CU, with each sequence provided as the unit circular or linear form. The relative amounts of wild-type and mutant satellite RNA sequence recovered from progeny virions reflected their relative abundances in the inoculum without regard to whether the sequence was supplied as a linear or a circular molecule. These results are consistent with models for the replication of the satellite RNA in which a circular form of the satellite RNA is a template for rolling circle transcription or is otherwise a replication intermediate or is readily converted to an intermediate. We also show that a circular form of a nonaccumulating satellite RNA mutant induced an increase in a satellite RNA that is endogenous to some tobacco ringspot virus virion preparations, as demonstrated previously for the linear form.

Base Sequence↗
PubMed1994

The antiviral activity of RNA-dye combinations.

The results of our previous studies (Jamison et al. 1988, 1989, 1990 a, b, c, d, e) have shown that the ability of intercalative dyes to modulate the antiviral activity of poly r(A-U) is related to the groove through which the dyes intercalate into the poly r(A-U). When poly r(A-U) is combined with the minor groove intercalating dyes or the minor/major groove intercalating dyes, optimum enhancement of antiviral activity is observed at the dye/ribonucleotide ratio predicted by the neighbor exclusion model (usually 1/4 or 1/6). No enhancement is observed when poly r(A-U) is combined with major groove intercalating dyes. When poly r(A-U) is combined with additional intercalative dyes to produce a dye/ribonucleotide ratio of 1/4 and a ribonucleotide concentration of 200 microM, the antiviral activity of poly r(A-U) is enhanced 8- to 20-fold, while 50% effective doses of the poly r(A-U) and the dyes decreases 18- to 347-fold. Interferon neutralization assays demonstrate that the interferon-inducing capability of the dye/poly r(A-U) combinations approximates the sum of the interferon-inducing capabilities of the poly r(A-U) and the dyes employed and suggests that the dyes potentiate the antiviral activity of poly r(A-U) without affecting the amount of interferon induced. Direct viral inactivation studies demonstrate that the dyes, poly r(A-U), and the dye/poly r(A-U) combinations do not inactivate VSV at concentrations near the 50% viral inhibitory dose. Assessment of cytotoxicity by microscope examination of HSF cell morphology and trypan blue exclusion indicates that the dye/poly r(A-U) combinations exhibit antiviral activity at concentrations well below those that induce cyto-toxicity. Several of the dyes and the dye/poly r(A-U) combinations exhibit anti-HIV-1 activity, suggesting that the enhancement phenomenon is not virus-specific nor host cell-specific. The enhancement phenomenon is sensitive to the base sequence of the polynucleotide with dye/poly r(A-U) and dye/poly r(G-C) combinations displaying enhanced antiviral activity, while dye/poly (rI).poly (rC) and dye/poly d(A-T) combinations do not. These results suggest that while intercalation of the dye and interferon induction are necessary for enhanced antiviral activity, neither intercalation nor interferon induction alone is sufficient to potentiate the antiviral activity of polyribonucleotides.

Antiviral Agents↗
PubMed1990

A low molecular weight DNA polymerase from wheat embryos.

The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase CI) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase CI did not inhibit animal DNA polymerases alpha and beta or wheat DNA polymerase A, whereas wheat DNA polymerases CII and B were much less affected than the CI enzyme. Several properties of enzyme CI were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase CI while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase CI can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase beta. The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase beta, an enzyme believed to be involved in nuclear DNA repair.

Amino Acids↗
PubMed1981

RNA polymerase is required for DNA initiation in vitro.

We have previously reported in vitro complementation assays for chromosome initiation that enable dnaA and dnaC mutant extracts to synthesize DNA. To examine the role of RNA polymerase in chromosome initiation, inhibitors of the enzyme and anti-RNA polymerase antibody were used. Though rifampicin failed to efficiently inhibit ribonucleoside triphosphate polymerization under the assay conditions, both streptolydigin and anti-RNA polymerase antibody abolished ribonucleic acid synthesis completely. Antibody effectively inhibited chromosome initiation in the dnaA mutant based reaction but streptolydigin did not. Neither streptolydigin nor antibody affected the dnaC-dependent assay. It was concluded that RNA polymerase is required for initiation but not necessarily to polymerize a polyribonucleotide. A scheme for the sequence of initiation events is presented.

Aminoglycosides↗
PubMed1988

Changes in body temperature and vasopressin content of brain neurons, in pregnant and non-pregnant guinea pigs, during fevers produced by Poly I:Poly C.

The synthetic polyribonucleotide pyrogen Poly I:Poly C (800 micrograms.kg-1) was injected intramuscularly on alternate days into pregnant and non-pregnant female guinea pigs. Pregnant animals, close to term, had smaller fevers in response to the pyrogen than did non-pregnant animals. Repeated injections of the pyrogen caused sequentially smaller fevers for the first 3-4 injections, particularly in non-pregnant animals, and this appeared to be like the tolerance usually developed to repeated injections of endotoxin. Continued pyrogen injections then caused, in non-pregnant animals, fevers of increasing magnitude until the original fever levels were reached, whereas in pregnant guinea pigs the fever responses remained reduced until parturition. The development of tolerance was associated with an increase in immunoreactivity for arginine vasopressin (AVP) in some neurons in the medial part of the paraventricular nucleus, and in terminals in the lateral septum and amygdala similar to changes found in these areas at term of pregnancy. These observations raise the possibility that AVP in these regions may have a role in the development of tolerance to pyrogens, and further quantitative studies of the AVP content of, and release from, nerve terminals projecting to the limbic system seem warranted.

Animals↗
PubMed1985

A possible adenine nucleotide storage form in normal and ischaemic rat heart.

Perfusion of Langendorff rat hearts with [14C]adenosine yields an acid-insoluble, radioactive product whose concentration falls during ischaemia. The properties of the substance show that it is a polyribonucleotide. It is suggested that it may be mitochondrial poly A acting as a storage form of adenine nucleotides.

Adenine Nucleotides↗
PubMed1980

The antiviral activity of ribosomal polynucleotides against encephalomyocarditis virus infection of mice.

Intraperitoneal administration of ribosomal RNA (rRNA) was found to protect mice against subsequent lethal infection by encephalomyocarditis (EMC) virus without induction of detectable amounts of circulating interferon. The nature of this effect was examined in terms of the types of natural polyribonucleotides which could afford such protection. rRNA prepared from E. coli was slightly more effective than chicken liver rRNA which was, in turn, more effective than yeast rRNA. 5S ribosomal RNA was not effective, whereas the slightly smaller 4S transfer RNA was as good as E. coli rRNA, suggesting that molecular size is not the sole criterion for the protective effect. The separated 16S and 23S E. coli rRNAs where each as effective as the unfractionated RNA. Anti-viral activity was lost after complete hydrolysis with alkali and nucleoside monophosphates were also inactive. Digestion of rRNA with pancreatic ribonuclease greatly decreased its antiviral activity whereas digestion with T1 ribonuclease had no effect indicating that fairly short oligonucleotides, but not of random nucleotide sequence, are active components in the protection of mice against infection by EMC virus. In vitro, no antiviral effect against EMC virus infection was observed in treatment of L cells under various conditions.

Animals↗
PubMedDate not supplied

A model for the coevolution of the genetic code and the process of protein synthesis: Review and assessment.

The contemporary genetic code and the process of protein biosynthesis most assuredly evolved from a simpler code and process. We believe that there was obligatory coevolution of the two and that the earlier code and process must have involved a more direct linkage between the amino acids and the information macromolecule. We propose that an early form of translating existed in which amino acids were attached directly to the 'messenger' RNA along the backbone as 2'OH aminoacyl esters. These esters then condensed with each other on the RNA backbone yielding a peptide covalently attached to the RNA, without the use of tRNA's and ribosomes. THis presentation is concerned with experimental data which indicate that such a simple translation system is possible and must have involved the following steps: (1) formation of the aminoacyl adenylate anhydride, (2) transfer of the amino acid from the adenylate to immidazole, (3) transfer of the amino acid from imidazole to 2'OH groups along the backbone of RNAs, (4) condensation of the amino acids to yield peptides. Steps (1)-(3) have been confirmed in chemical systems. Our preliminary evidence indicates step (4) is also possible. The aminoacylation of polyribonucleotides and the subsequent formation of peptides is a dynamic and experimentally accessible system for studying genetic coding specfities and our present studies are now concentrated on step (4), looking for such specifities.

Acylation↗
PubMed1992

Experimental studies on the origin of the genetic code and the process of protein synthesis: a review update.

This article is an update of our earlier review (Lacey and Mullins, 1983) in this journal on the origin of the genetic code and the process of protein synthesis. It is our intent to discuss only experimental evidence published since then although there is the necessity to mention the old enough to place the new in context. We do not include theoretical nor hypothetical treatments of the code or protein synthesis. Relevant data regarding the evolution of tRNAs and the recognition of tRNAs by aminoacyl-tRNA-synthetases are discussed. Our present belief is that the code arose based on a core of early assignments which were made on a physico-chemical and anticodonic basis and this was expanded with new assignments later. These late assignments do not necessarily show an amino acid-anticodon relatedness. In spite of the fact that most data suggest a code origin based on amino acid-anticodon relationships, some new data suggesting preferential binding of Arg to its codons are discussed. While information regarding coding is not increasing very rapidly, information regarding the basic chemistry of the process of protein synthesis has increased significantly, principally relating to aminoacylation of mono- and polyribonucleotides. Included in those studies are several which show stereoselective reactions of L-amino acids with nucleotides having D-sugars. Hydrophobic interactions definitely play a role in the preferences which have been observed.

5'-Nucleotidase↗
PubMed1984

Binding constants of phenylalanine for the four mononucleotides.

Earlier work has shown that several properties of amino acids correlate directly with properties of their anticodonic nucleotides. Furthermore, in precipitation studies with thermal proteinoids and homopolyribonucleotides, an anticodonic preference was displayed between Lys-rich, Pro-rich and Gly-rich thermal proteinoids and their anticodonic polyribonucleotides. However, Phe-rich thermal proteinoid displayed a preference for its codonic nucleotide, poly U. This inconsistency seemed to be explained by a folding in of the hydrophobic residues of Phe causing the proteinoid to appear more hydrophilic. The present work used nuclear magnetic resonance techniques to resolve a limited question: To which of the four nucleotides does Phe bind most strongly? The results show quite clearly that Phe binds most strongly to its anticodonic nucleotide, AMP.

Adenosine Monophosphate↗
PubMed1998

Human extracellular ribonucleases: multiplicity, molecular diversity and catalytic properties of the major RNase types.

Human extracellular ribonucleases (RNase), together with other members of the mammalian RNase A superfamily, can be classified into four different RNase families on the basis of their structural, catalytic and/or biological properties. Their occurrence and main distinctive features have been described, and the information available on their catalytic properties has been analysed and discussed in comparison with those of other animal RNases. On the basis of some results obtained with various single- and double-stranded polyribonucleotides, it has been proposed that while pancreatic-type (pt) RNases could be defined as single-strand/pyrimidine 'preferring' ribonucleases, mammalian nonpancreatic-type (npt) RNases may be referred to as single-strand/pyrimidine 'specific' ribonucleases. In addition, some data concerning human nptRNases may support the suggestion [Cuchillo et al. (1993) FEBS Lett. 333: 207-210] that the enzyme 'ribonuclease' should be reclassified as 'transferase'.

Amino Acid Sequence↗
PubMed1975

Modified nucleotide polymers as inhibitors of DNA polymerases.

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.

Cell Line↗
PubMed1975

High molecular weight deoxyribonucleic acid polymerase of LF hepatoma. Purification and properties.

A high molecular weight DNA polymerase has been purified from the cytosol of a fast growing hepatoma: LF hepatoma. This enzyme sediments at 11.3 S under polymerization reaction conditions (6 mM KCl) and at 8.3 S in higher salt concentrations (200 mM KCl). In either case, no activity is seen in the 3 to 4 S region where low molecular weight DNA polymerase is found. The purified enzyme has a neutral pH optimum and requires a divalent cation, all four deoxyribonucleoside triphosphates and an initiated DNA template for maximal activity. The synthetic template specificity of LF DNA polymerase has been studied. Although this enzyme cannot copy a polyribonucleotide template, the ribostrand of a synthetic hybrid can be used with low efficiency as an initiator for the synthesis of the complementary deoxyribonucleotide strand. The activity of the purified enzyme is strongly inhibited by thiol-blocking agents. The general properties of LF DNA polymerase are similar to those of high molecular weight mammalian DNA polymerases. In our experimental conditions, the error frequency of this tumoral DNA polymerase was no greater than that made by the purified high molecular weight DNA polymerase of regenerating rat liver.

Animals↗
PubMed1976

Metal-polynucleotide interactions. A comparison of carcinogenic and non-carcinogenic metals in vitro.

The effects of divalent cations on mixing curves of synthetic polyribonucleotides in solution are described. Manganese and cadmium chlorides at 10(-3) M induce changes suggestive of base mispairing. MnCl2 induces mispairing in complexes formed between both poly(I) and poly(C,U) and poly(I) and poly(C,A) while CdCl2 affects base pairing between poly(I) and poly(C,U) only. By contrast, the chlorides of magnesium and zinc show no mispairing effects with either polymer pair. Manganese and cadmium are both reported carcinogens in animals while magnesium and zinc are not. The possibility that direct metal-nucleic acid interaction may be involved in metal carcinogenesis is discussed.

Binding Sites↗
PubMed1976

Polyadenylate polymerase from cytoplasm and nuclei of N.I.H.-Swiss mouse embryos.

Poly (A) polymerase activity from cytoplasm and nuclei of 12-16-day-old mouse embryos has been partially purified by (NH4)2SO4 fractionation, DEAE-cellulose, phosphocellulose and tRNA-Sepharose affinity chromatography, and their properties have been compared. The nuclear and cytoplasmic enzymes exhibit similar chromatographic elution profiles, and similar biochemical and physical properties. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP and an oligo- or polyribonucleotide primer. With tRNA, the divalent salt concentrations for optimum enzyme activity are 1 mM MnCl2 or 10 mM MgCl2. The enzyme activity with MnCl2 is 10-15-fold higher than that with MgCl2. The molecular weight of the native enzyme is about 65 000 and its sedimentation coefficient is around 4.5 S. The average chain length synthesized by the enzyme is between 10 and 13 nucleotides. The inhibitors of RNA polymerase do not affect poly (A) polymerase activity; however, some synthetic rifamycin SV derivatives are potent inhibitors of this enzyme.

Animals↗
PubMed1977

Differential, structure-dependent susceptibility of poly(A) and RNA to monomeric and dimeric pancreatic ribonuclease A.

Cross-linked dimers of bovine RNAase A are definitely more efficient than monomers at degrading polyadenylic acid under conditions of ionic strength and pH, where the polymer assumes either a double-helical or an ordered single-stranded, base-stacked structure. The opposite occurs, i.e., monomers of RNAase A are definitely more active than dimers,when poly(A) is digested by the two enzyme species under conditions where the conformation of the polymer is essentially that of a random coil. The same pattern of events occurs when total RNA from Escherichia coli or single-stranded RNA of f2 sus11 bacteriophage are used as substrates under opposite ionic-strength conditions. In the presence of high salt concentrations, favouring the formation and the stability of a secondary structure in self-complementary sequences of RNA, the ribonucleic acids are degraded at a higher rate by dimers than by monomers of bovine RNAase A. The opposite occurs in the presence of very low salt concentrations, i.e. when the RNAs are in solution presumably as random coils. These observations are discussed in the light of a hypothesis already advanced to understand the mechanism of enzymic degradation of secondary structures of polyribonucleotides.

Animals↗