DNA adsorption and release from cat-anionic vesicles made of sodium dodecylsulfate-dodecyldimethylammonium bromide (SDS-DDAB) in nonstoichiometric amounts was investigated by different electrochemical, spectroscopic, and biomolecular strategies. The characterization of the vesicular system was performed by dynamic light scattering, which allowed estimating both its size and distribution function(s). The interaction dynamics was followed by dielectric spectroscopy and zeta-potential, as well as by agarose gel electrophoresis, AGE. Also, circular dichroism, CD, measurements were carried out, to ascertain possible structural rearrangements of DNA, consequent to the interactions with the cat-anionic vesicles. CD demonstrates that vesicle-bound DNA retains its native conformation. The results obtained by the aforementioned techniques are consistent and indicate that binding saturation is obtained at a [DNA/vesicles] charge ratio close to 0.8, considering only the excess surface charges on the vesicles. This result is apparently in contradiction with a purely electrostatic approach and is tentatively ascribed to the distance between charges on the biopolymer and the vesicle surface, respectively. A possible interpretation is discussed. The nucleic acid can be completely retrieved from the vesicles upon addition of adequate amounts of SDS, which is the defective surfactant in the vesicular system. Precipitation of the poorly soluble SD-DDA salt results in an almost complete release of DNA.

Initial rate kinetics of the formation of ternary complexes of Escherichia coli 30S ribosomal subunits, poly(uridylic acid), and N-acetylphenylalanyl transfer ribonucleic acid in the presence and in the absence of IF-3 are consistent with the hypothesis that the ternary complex is formed through a random order of addition of polynucleotide and aminoacyl-tRNA to separate and independent binding sites on the 30S ribosomes. The transformation of an intermediate into a stable ternary complex which probably entails a rearrangement of the ribosome structure leading to a codon-anticodon interaction represents the rate-limiting step in the formation of the ternary complex. The rate constant of this transformation, as well as the association constants for the formation of the 30S-poly(U) and 30S-N-AcPhe-tRNA binary complexes, are enhanced by the presence of IF-3 which acts as a kinetic effector on reactions which are intrinsic properties of the 30S ribosome. The IF-3-induced modification of these kinetic parameters of the 30S ribosomal subunit can per se explain the effect of IF-3 on protein synthesis without invoking a specific action at the level of the mRNA-ribosome interaction. This seems to be confirmed by the finding that IF-3 can stimulate several-fold the formation of a ternary complex even if one by-passes the ribosome-template binding step by starting with a covalent 30S-polynucleotide binary complex. Furthermore, the above-mentioned changes induced by IF-3 appear to be compatible with the previously proposed idea that the binding of the factor modifies the conformation of the 30S subunit. The random order of addition of substrates determined for the 30S-N-AcPhe-tRNA-poly(U) model system was found to be valid also for the more physiological 30S initiation complex containing poly(A,U.G) and (fMet-tRNA formed at low Mg2+ concentration in the presence of GTP and all three initiation factors.

In higher eukaryotes, increasing evidence suggests, gene expression is to a large degree controlled by RNA. Regulatory RNAs have been implicated in the management of neuronal function and plasticity in mammalian brains. However, much of the molecular-mechanistic framework that enables neuronal regulatory RNAs to control gene expression remains poorly understood. Here, we establish molecular mechanisms that underlie the regulatory capacity of neuronal BC RNAs in the translational control of gene expression. We report that regulatory BC RNAs employ a two-pronged approach in translational control. One of two distinct repression mechanisms is mediated by C-loop motifs in BC RNA 3 stem-loop domains. These C-loops bind to eIF4B and prevent the factor's interaction with 18S rRNA of the small ribosomal subunit. In the second mechanism, the central A-rich domains of BC RNAs target eIF4A, specifically inhibiting its RNA helicase activity. Thus, BC RNAs repress translation initiation in a bimodal mechanistic approach. As BC RNA functionality has evolved independently in rodent and primate lineages, our data suggest that BC RNA translational control was necessitated and implemented during mammalian phylogenetic development of complex neural systems.Translational control is an important means for the regulation of gene expression in eukaryotic cells (22). In neurons, the local translation of select mRNAs in synaptodendritic domains is considered a key determinant of neuronal function and plasticity (11,13,28,42). Strict control of local translation is essential to ensure that relevant proteins are synthesized only when and where needed (11). Progress has been made over the last 10 years as translational control mechanisms have been investigated in neurons, and several translational regulators have been identified (33,46). In one of these mechanisms, the effectors of neuronal translational control are regulatory BC RNAs (2,8,(43)(44)(45).Dendritic BC RNAs, neuronal small cytoplasmic RNAs (scRNAs) that include rodent BC1 RNA and primate BC200 RNA (20,21,40,41), are non-protein-coding RNAs that regulate translation at the level of initiation (43, 44). Translational control mediated by BC1 RNA is important in the management of neuronal excitability (8,47,48). Lack of BC1 RNA in a BC1Ϫ/Ϫ animal model triggers increased group I metabotropic glutamate receptor-dependent synthesis of select synaptic proteins (47). Such alterations in the absence of BC1 RNA precipitate neuronal metabotropic glutamate receptor-mediated hyperexcitability that manifests in the form of exaggerated cortical gamma frequency oscillations, epileptogenic neuronal responses, and generalized seizures triggered by auditory stimulation (47, 48). These phenotypical manifestations are consonant with the molecular role of BC RNAs as translational repressors. BC1 RNA inhibits recruitment of the 43S preinitiation complex to the mRNA (44), a rate-limiting step in translation initiation that is mediated by the eIF4 family of eukaryotic initiation factors (6,9,12,31).The...

Initiation factor IF-3 causes the destabilization of preformed ternary complexes of 30-S ribosomal subunit, codons and aminoacyl-tRNAs or peptidyl-tRNA. This destabilization is dilutiondependent and affects all ternary complexes with the exception of those containing the initiator Wet-tRNA, which remain more resistant to IF-3-induced destabilization under the various conditions studied. Several possible reasons for this specificity have been examined. It was found that the basis for the specificity is not: (a) an intrinsic greater stability of the ternary complexes containing met-tRNA, (b) the amount of aminoacyl-tRNA bound to the ribosome, (c) the conditions under which the ternary complex is made or (d) the formylation of the amino group. On the other hand, the nature of the polynucleotide in response to which the ternary complex is formed was found to influence the amount of aminoacyl-tRNA bound to the ribosome, and to some extent the amount of aminoacyl-tRNA which can be released. The ternary complex containing the mischarged initiator tRNA fVal-tRNAfMet displays greater resistance to the IF-3-induced destabilization than the complex containing fVal-tRNAVa'. These results indicate that the specificity of the IF-3 activity is due to the special structural feature of the initiator tRNA molecule and to some extent to the nature of the polynucleotide. The IF-3-induced destabilization of ternary complexes was found to be little affected by variations in reaction conditions, so that this IF-3 activity can be used to measure the stoichiometric binding of IF-3 to the ribosome over a broad range of pH and K + and M$+ concentrations. Several antibiotics have been tested for their capacity to interfere with this reaction ; only high concentrations of tetracycline blocked this IF-3 activity.The IF-3-induced destabilization of the 30-Ssubunit . codon . aminoacyl-tRNA ternary complex [l, 21 results from the stoichiometric binding of one IF-3 molecule per 30-S particle setting off a conformational transition of the ribosomal subunit [2 -41. This IF-3 property can form the basis for an alternative approach to the study of the interaction between ribosomes and IF-3 in place of the assay involving sucrose gradient centrifugation [5 -71, provided that the specificity as well as the conditions influencing this reaction are well characterized. In addition, a better understanding of the molecular mechanism by which this peculiar IF-3 activity is brought about should prove useful in solving the more general mechanism by which IF-3 exercises its physiological activity at the ribosomal level.In this paper the basis for the specificity of the IF-3-induced destabilization of ternary complexes containing initiator or non-initiator aminoacyl-t RNAs (aa-tRNAs) as well as the effect of varying the reaction conditions (pH, ionic concentration, etc.) 203-208 (1970).DeJnifion. A,,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-cm pathlength cell. MATERIALS AND M...

The interactions between cat-anionic (an acronym indicating surfactant aggregates (micelles and vesicles) formed upon mixing cationic and anionic surfactants in nonstoichiometric amounts) vesicles and DNA have been the subject of intensive studies because of their potential applications in biomedicine. Here we report on the interactions between DNA and cetyltrimethylammonium bromide (CTAB)-sodium octyl sulfate (SOS) cat-anionic vesicles. The study was performed by combining dielectric relaxation spectroscopy, circular dichroism, dynamic light scattering, ion conductivity, and molecular biology techniques. DNA is added to positively charged vesicles until complete charge neutralization of the complex and formation of lipoplexes. This occurs when the mole ratio between the phosphate groups of DNA and positive charges on the vesicle is about 1.8. Above this threshold the nucleic acid in excess remains free in solution. This very interesting new result shows that anionic surfactants are not expelled upon saturation, and therefore, no formation of micelles occurs. Furthermore, vesicle-bound DNA can be released in its native form, as confirmed by dielectric spectroscopy and circular dichroism measurements. The nucleic acid is released upon addition of SOS, which competes with the phosphate groups of the DNA: this results in the demolition of the CTAB-SOS cat-anionic vesicles. These results indicate the possibility of a controlled DNA release and might be of interest in biomedicine.

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Background: Resveratrol is a non flavonoid polyphenol compound present in many plants and fruits and, at especially high concentrations, in the grape berries of Vitis vinifera. This compound has a strong bioactivity and its cytoprotective action has been demonstrated, however at high concentrations the drug exhibits also an effective anti-proliferative action. We recently showed its ability to abolish the effects of oxidative stress in cultured cells. In this work we assayed the bioactivity of resveratrol as antiproliferative and antiviral drug in cultured fibroblasts. Studies by other Authors showed that this natural compound inhibits the proliferation of different viruses such as herpes simplex, varicella-zoster and influenza A. The results presented here show an evident toxic activity of the drug at high concentrations, on the other hand at sub-cytotoxic concentrations, resveratrol can effectively inhibit the synthesis of polyomavirus DNA. A possible interpretation is that, due to the damage caused by resveratrol to the plasma membrane, the transfer of the virus from the endoplasmic reticulum to the nucleus, may be hindered thus inhibiting the production of viral DNA.

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