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PubMed2005

Electrostatic potentials of E.coli genome DNA.

Distribution of electrostatic potential of the complete sequence of E. coli genome was calculated. Comparative analysis of electrostatic patterns for 359 promoter and nonpromoter nucleotide sequences was carried out. It is found that nonpromoter regions are characterized by more homogeneous distribution of electrostatic potential with no common specific elements. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which may be involved as promoter signal elements in RNA-polymerase-promoter recognition.

Base Sequence↗
PubMed2005

Choosing between DNA and RNA: the polymer specificity of RNA helicase NPH-II.

NPH-II is a prototypical member of the DExH/D subgroup of superfamily II helicases. It exhibits robust RNA helicase activity, and a detailed kinetic framework for unwinding has been established. However, like most SF2 helicases, there is little known about its mode of substrate recognition and its ability to differentiate between RNA and DNA substrates. Here, we employ a series of chimeric RNA-DNA substrates to explore the molecular determinants for NPH-II specificity on RNA and to determine if there are conditions under which DNA is a substrate. We show that efficient RNA helicase activity depends exclusively on ribose moieties in the loading strand and in a specific section of the 3'-overhang. However, we also document the presence of trace activity on DNA polymers, showing that DNA can be unwound under extremely permissive conditions that favor electrostatic binding. Thus, while polymer-specific SF2 helicases control substrate recognition through specific interactions with the loading strand, alternative specificities can arise under appropriate reaction conditions.

Animals↗
PubMed2002

Molecular modeling approaches for determining gene function: application to a putative poly-A binding protein from Leishmania amazonensis (LaPABP).

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 amino acids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

Animals↗
PubMed1999

[Catalytic properties of recombinant pancreatic ribonuclease A-K7H].

Pancreatic ribonuclease A is an enzyme that binds up ribonucleic acid (RNA) along the multiple binding subsites that essentially recognize the negatively charged phosphates of the substrate. It is endoribonuclease that catalyse depolimerization of single-stranded RNA. This work gives additional support to the existence of the phosphate-binding site p2 and confirms the central role of Lys-7 in establishing and electrostatic interraction with a phosphate group of the substrate. In this work catalytic properties of recombinant ribonuclease K7H have been studied. This enzyme is a mutant enzyme which contains histidine instead of lysine in a position 7, amino-acid that participates in the main catalytic center of RNase A, named p1. It was obtained by site-directed mutagenesis. Kinetic parameters of K7H have determined with C > p i poli (C) as substrates at pH 5.5 i 7.5. Kinetic parameters of K7H for C > p and as a substrate at pH 5.5 have not altered, but at pH 7.5 were significantly increased. Value Km was also increased, that indicates decreasing of affinity. Increasing of catalysis was double. Results of kinetic parameters of K7H with poli (C) as a substrate in pH 5.5 have shown slight difference according to kinetic parameters of commercial RNase A with poli (C). Significant decreasing of values of all kinetic parameters for K7H were reaction at pH 7.5.

Catalysis↗
PubMed1999

Deguanylation of human immunodeficiency virus (HIV-1) RNA by recombinant pokeweed antiviral protein.

Modeling studies, combined with the molecular docking of the trinucleotide GGG into the active site of the deadenylating RNA N-glycosidase pokeweed antiviral protein (PAP), indicated that a guanine base can fit into the active site pocket of PAP without disturbing its unique geometry and is sandwiched between residues Tyr(72) and Tyr(123) very much like an adenine base. The guanine base can form two specific hydrogen bonds with the active site residues Ser(121) and Val(73) and the attached negatively charged phosphate groups can entertain stabilizing electrostatic interactions with two clusters of positively charged patches on the PAP surface formed by Lys(210) and Arg(179) from one side and Arg(122) and Arg(135) from the other side of the active site. These observations prompted the hypothesis that the RNA depurinating activity of PAP may not be restricted to adenine residues and PAP should be capable of deguanylating ribosomal and viral RNA as well. This hypothesis was experimentally confirmed by direct demonstration that guanine base is released from both ribosomal and HIV-1 RNA after treatment with purified recombinant PAP using quantitative high performance liquid chromatography. Recombinant PAP released adenine and guanine residues at a 1:1 ratio from HIV-1 RNA and at an approximately 3:1 (adenine:guanine) ratio from Escherichia coli ribosomal RNA. At a concentration of 5 microM, recombinant PAP released 263 +/- 10 pmol of adenine and 100 +/- 11 pmol of guanine from 1 microgram of E. coli ribosomal RNA (16S + 23S) within 4 h of treatment. By comparison, 138 +/- 12 pmol of adenine and 143 +/- 10 pmol of guanine were released from 1 microgram of HIV-1 RNA under identical treatment conditions (5 microM recombinant PAP, 4 h treatment). The deguanylation of the ribosomal and viral RNA targets by recombinant PAP was concentration-dependent and is abolished by alanine substitutions of the catalytic active site residues Tyr(72) and Tyr(123). To our knowledge, these findings provide the first evidence that PAP can deguanylate both ribosomal and viral RNA.

Adenine Nucleotides↗
PubMed2001

Antibody masking renders HIV-1 resistant to cationic membrane filtration through alteration of its electrostatic characteristics.

Previously, it was demonstrated that any human immunodeficiency virus type 1 (HIV-1) strain proliferating in peripheral blood mononuclear cells (PBMCs) in vitro, and resuspended in seronegative plasma, could be captured efficiently (mean > 95%) by a porous polypropylene (PP) membrane modified cationically. We investigated if this cationic membrane could capture HIV-1 obtained from seropositive plasma, and confirmed whether this membrane was effective for the preparation of safe plasma products against HIV-1 transmission. Thirty-six seropositive plasma samples derived from HIV-1 positive cohorts in New York and Lusaka (Republic of Zambia), including 18 cases of acquired immunodeficiency syndrome (AIDS) related complex, AIDS and five terminal cases of AIDS, were filtered through the cationic membrane to determine the reduction of RNA concentration, the gag p24 concentration, and infectious titer. Only a small reduction in RNA concentration (mean < 20%) and almost no decrease in gag concentration (mean < 2%) were obtained, despite the fact that the infectivity was eliminated entirely by the filtration. Due to the possibility that anti-HIV-1 antibodies in patients' plasma combine with HIV-1, laboratory-adapted HIV-1(HTLV-IIIB) was mixed with seropositive plasma to test the effect of antibodies on HIV-1 adsorption, and also to investigate the interfacial electrokinetic potential (zeta-potential) of both intact and plasma-treated HIV-1. The zeta-potential of HIV-1(HTLV-IIIB) in the presence of seropositive plasma was neutral as opposed to negative when stored in seronegative plasma or culture medium. Also the rate of HIV-1 capture by the membrane, as determined by the reduction in RNA concentration, sank from 95% to 20%, the same capture percentage observed when filtering plasma of patients. These findings suggested that in patients' plasma, the antibody-masked HIV-1 comprise most of the viral population, and was not trapped on the cationic membrane because of its electrostatic character. Conversely, the cationic membrane was thought to adsorb antibody-free HIV-1 exclusively. It was suggested that each viral swarm had its own zeta-potential, and this difference in electrostatic character determined the extent of the viral adsorption by the cationic membrane.

Cations↗
PubMed2000

Structure of the Sm binding site from human U4 snRNA derived from a 3 ns PME molecular dynamics simulation.

A molecular dynamics simulation of the Sm binding site from human U4 snRNA was undertaken to determine the conformational flexibility of this region and to identify RNA conformations that were important for binding of the Sm proteins. The RNA was fully-solvated (>9,000 water molecules) and charge neutralized by inclusion of potassium ions. A three nanosecond MD simulation was conducted using AMBER with long-range electrostatic forces considered using the particle mesh Ewald summation method. The initial model of the Sm binding site region had the central and 3' stem-loops that flanked the Sm site co-axial with one another, and with the single-stranded Sm binding site region ([I] conformation). During the course of the trajectory, the axes of the 3' stem-loop, and later the central stem-loop, became roughly orthogonal from their original anti-parallel orientation. As these conformational changes occurred, the snRNA adopted first an [L] conformation, and finally a [U] conformation. The [U] conformation was more stable than either the [I] or [L] conformations, and persisted for the final 1 ns of the trajectory. Analysis of the structure resulting from the MD simulations revealed the bulged nucleotide, U114, and the mismatched Ag91-G110 base pair provided distinctive structural features that may enhance Sm protein binding. Based on the results of the MD simulation and the available experimental data, we proposed a mechanism for the binding of the Sm protein sub-complexes to the snRNA. In this model, the D1/D2 and E/F/G Sm protein sub-complexes first bind the snRNA in the [U] conformation, followed by conformational re-arrangement to the [I] conformation and binding of the D3/B Sm protein sub-complex.

Base Sequence↗
PubMed1994

On the role of lipids in cell regulatory economy.

Prenylated proteins, which contain a lipid tail, may function to enhance the self-assembly of complementary molecular structures by providing a monomolecular film between the two combining regions so that electrostatic forces of attraction are greatly increased over that which would be established with water molecules in the intervening spaces. Such interactions may be important in DNA regulation, RNA processing, and a variety of protein-protein interactions.

Cell Physiological Phenomena↗
PubMed2000

Large conformational changes in the maturation of a simple RNA virus, nudaurelia capensis omega virus (NomegaV).

An assembly intermediate of a small, non-enveloped RNA virus has been discovered that exhibits striking differences from the mature virion. Virus-like particles (VLPs) of Nudaurelia capensis omega virus (NomegaV), a T=4 icosahedral virus infecting Lepidoptera insects, were produced in insect cells using a baculovirus vector expressing the coat protein. A procapsid form was discovered when NomegaV VLPs were purified at neutral pH conditions. These VLPs were fragile and did not undergo the autoproteolytic maturation that occurs in the infectious virus. Electron cryo-microscopy (cryoEM) and image analysis showed that, compared with the native virion, the VLPs were 16% larger in diameter, more rounded, porous, and contained an additional internal domain. Upon lowering the pH to 5.0, the VLP capsids became structurally indistinguishable from the authentic virion and the subunits autoproteolyzed. The NomegaV protein subunit coordinates, which were previously determined crystallographically, were modelled into the 28 A resolution cryoEM map of the procapsid. The resulting pseudo-atomic model of the NomegaV procapsid demonstrated the large rearrangements in quaternary and tertiary structure needed for the maturation of the VLPs and presumably of the virus. Based on this model, we propose that electrostatically driven rearrangements of interior helical regions are responsible for the large conformational change. These results are surprising because large structural rearrangements have not been found in the maturation of any other small RNA viruses. However, similarities of this conformational change to the maturational processes of more complex DNA viruses (e.g. bacteriophages and herpesvirus) and to the swelling of simple plant viruses suggest that structural changes in icosahedral viruses, which are integral to their function, have similar strategies and perhaps mechanisms.

Animals↗
PubMed1984

Effects of DEAE-dextran on infection and hemolysis by VSV. Evidence that nonspecific electrostatic interactions mediate effective binding of VSV to cells.

The polycation DEAE-dextran increased the binding of VSV to BHK cells about fourfold over a wide range of VSV concentrations. The same proportion of bound virions was internalized by the cell in the presence or absence of DEAE-dextran. Viral primary RNA production was increased to the equivalent of a 4- to 4.5-fold increase in multiplicity of infection in the presence of DEAE-dextran, closely paralleling the increase in total VSV binding to the cell. Viral secondary RNA production was increased only to the equivalent of about twofold increase in multiplicity. The kinetics of both primary and secondary RNA production were indistinguishable in the presence or absence of DEAE-dextran. DEAE-dextran had to be present simultaneously with the input virions in order to enhance RNA production; addition even 30 min after infection was ineffective. Addition of the polycation DEAE-dextran was also required for appreciable VSV-induced hemolysis of human erythrocytes; erythrocytes of several other species were hemolyzed in the absence of DEAE-dextran, but hemolysis was enhanced by its presence. Maximal binding and hemolysis occurred at pH 5.0 and 37 degrees. Vesicles containing only G protein and viral lipid were 40% as hemolytic as intact virions at pH 5.0, but were inactive at pH 6.0; "spikeless" virions lacking G, or protein-free viral lipid vesicles were not hemolytic, showing that G protein is necessary for hemolysis. These results, together with other recent observations, suggest that multiple electrostatic interactions between VSV and the cell surface, rather than an affinity for specific surface molecules, mediated the productive (infection producing) binding of VSV to the cell surface.

Animals↗
PubMed2001

Solution structure of HI0257, a bacterial ribosome binding protein.

A novel bacterial ribosome binding protein, protein Y (also known as YfiA), was recently shown to reside at the 30S/50S subunit interface and to stabilize the ribosomal 70S complex against dissociation at low magnesium ion concentrations. We report here the three-dimensional NMR structure in solution of a homologue from Haemophilus influenzae, HI0257, that has 64% sequence identity to protein Y. The 107 residue protein has a beta-alpha-beta-beta-beta-alpha folding topology with two parallel alpha-helices packed against the same side of a four-stranded beta-sheet. The closest structural relatives are proteins with the double-stranded RNA-binding domain (dsRBD) motif although there is little (<10%) sequence homology. The most immediate differences between the dsRBD and HI0257 structures are that (1) HI0257 has a larger beta-sheet motif with an extra beta-strand at the N-terminus, (2) the helices are parallel in HI0257 but at an angle of about 30 degrees to each other in the dsRBD, and (3) HI0257 lacks the extended loop commonly seen between the first and second beta-strands of the dsRBD. Further, an analysis of the surface electrostatic potential in HI0257 and the dsRBD family reveals significant differences in the location of contiguous positively (and negatively) charged regions. The structural data, in combination with sequence analysis of HI0257 and its homologues, suggest that the most likely mode of RNA recognition for HI0257 may be distinct from that of the dsRBD family of proteins.

Amino Acid Sequence↗
PubMed1998

A 'retro-inverso' PNA: structural implications for DNA and RNA binding.

'Retro-inverso' peptide nucleic acid (PNA) monomers of thymine (T*: N-(amidomethyl)-N-(N1-thyminyl-acetyl)-beta-alanyl) (and adenine) have been prepared and introduced in PNA oligomers. A homo 'retro-inverso' T*8 PNA was found not to hybridize to a complementary DNA or RNA oligonucleotide, whereas introduction of one retro-inverso thymine unit into the middle of a normal PNA 15-mer resulted in a c.a. 8 degrees C destabilization of the complex of this oligomer with a complementary DNA or RNA oligomer. In an effort to compensate for the structural nucleobase 'phase-shift' caused by the T* monomer by also introducing a beta-alanine monomer it is concluded that the effect of the T* backbone is -7 degrees C when hybridizing to DNA and -4.5 degrees C when hybridizing to RNA. Nonetheless, the T* unit shows good sequence discrimination comparable to that of normal PNA. Molecular dynamics simulations indicate an unfavourable conformation of the backbone amide carbonyl group resulting in reduced interaction with the aqueous medium and an 'electrostatic clash' with the carbonyl of the nucleobase linker. These results show that a simple inversion of an amide bond in the PNA backbone has a dramatic, and hardly predictable, effect on the DNA mimicking properties of the oligomer.

Crystallography, X-Ray↗
PubMed2005

Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids.

An analogous series of cationic lipids (1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA) and 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA)) possessing 0, 1, 2 or 3 double bonds per alkyl chain respectively, was synthesized to determine the correlation between lipid saturation, fusogenicity and efficiency of intracellular nucleic acid delivery. 31P-NMR analysis suggests that as saturation increases, from 2 to 0 double bonds, lamellar (L(alpha)) to reversed hexagonal (H(II)) phase transition temperature increases, indicating decreasing fusogenicity. This trend is largely reflected by the efficiency of gene silencing observed in vitro when the lipids are formulated as Stable Nucleic Acid Lipid Particles (SNALPs) encapsulating small inhibitory RNA (siRNA). Uptake experiments suggest that despite their lower gene silencing efficiency, the less fusogenic particles are more readily internalized by cells. Microscopic visualization of fluorescently labelled siRNA uptake was supported by quantitative data acquired using radiolabelled preparations. Since electrostatic binding is a precursor to uptake, the pKa of each cationic lipid was determined. The results support a transfection model in which endosomal release, mediated by fusion with the endosomal membrane, results in cytoplasmic translocation of the nucleic acid payload.

Cations↗
PubMed2000

Structural equilibria in RNA as revealed by 19F NMR.

We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.

Fluorine↗
PubMed2006

Structural basis of glmS ribozyme activation by glucosamine-6-phosphate.

The glmS ribozyme is the only natural catalytic RNA known to require a small-molecule activator for catalysis. This catalytic RNA functions as a riboswitch, with activator-dependent RNA cleavage regulating glmS messenger RNA expression. We report crystal structures of the glmS ribozyme in precleavage states that are unliganded or bound to the competitive inhibitor glucose-6-phosphate and in the postcleavage state. All structures superimpose closely, revealing a remarkably rigid RNA that contains a preformed active and coenzyme-binding site. Unlike other riboswitches, the glmS ribozyme binds its activator in an open, solvent-accessible pocket. Our structures suggest that the amine group of the glmS ribozyme-bound coenzyme performs general acid-base and electrostatic catalysis.

5' Untranslated Regions↗
PubMed2003

Translocation properties of primitive molecular machines and their relevance to the structure of the genetic code.

We address the question, related with the origin of the genetic code, of why are there three bases per codon in the translation to protein process. As a follow-up to our previous work (Aldana et al., 1998, Martínez-Mekler et al., 1999a,b), we approach this problem by considering the translocation properties of primitive molecular machines, which capture basic features of ribosomal/messenger RNA interactions, while operating under prebiotic conditions. Our model consists of a short one-dimensional chain of charged particles (rRNA antecedent) interacting with a polymer (mRNA antecedent) via electrostatic forces. The chain is subject to external forcing that causes it to move along the polymer which is fixed in a quasi-one-dimensional geometry. Our numerical and analytic studies of statistical properties of random chain/polymer potentials suggest that, under very general conditions, a dynamics is attained in which the chain moves along the polymer in steps of three monomers. By adjusting the model in order to consider present-day genetic sequences, we show that the above property is enhanced for coding regions. Intergenic sequences display a behavior closer to the random situation. We argue that this dynamical property could be one of the underlying causes for the three-base codon structure of the genetic code

Animals↗
PubMed2006

In vitro-reassembled plant virus-like particles for loading of polyacids.

The coat protein (CP) of certain plant viruses may reassemble into empty virus-like particles (VLPs) and these protein cages may serve as potential drug delivery platforms. In this paper, the production of novel VLPs from the Hibiscus chlorotic ringspot virus (HCRSV) is reported and the capacity to load foreign materials was characterized. VLPs were readily produced by destabilizing the HCRSV in 8 M urea or Tris buffer pH 8, in the absence of calcium ions, followed by removal of viral RNA by ultrahigh-speed centrifugation and the reassembly of the CP in sodium acetate buffer pH 5. The loading of foreign materials into the VLPs was dependent on electrostatic interactions. Anionic polyacids, such as polystyrenesulfonic acid and polyacrylic acid, were successfully loaded but neutrally charged dextran molecules were not. The molecular-mass threshold for the polyacid cargo was about 13 kDa, due to the poor retention of smaller molecules, which readily diffused through the holes between the S domains present on the surface of the VLPs. These holes precluded the entry of large molecules, but allowed smaller molecules to enter or exit. The polyacid-loaded VLPs had comparable size, morphology and surface-charge density to the native HCRSV, and the amount of polyacids loaded was comparable to the weight of the native genomic materials. The conditions applied to disassembly-reassembly of the virions did not change the structural conformation of the CP. HCRSV-derived VLPs may provide a promising nano-sized protein cage for delivery of anionic drug molecules.

Acids↗