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Results for “RNA electrostatics”

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At least 145 records · Page 8Linked to original sources

PubMed2006

Thermodynamics of the fragile X mental retardation protein RGG box interactions with G quartet forming RNA.

Fragile X syndrome, the most common form of inherited mental retardation, is the result of an unstable expansion of a CGG trinucleotide repeat in the 5' UTR of the fragile X mental retardation-1 (FMR1) gene. The abnormal hypermethylation of the expanded CGG repeats causes the transcriptional silencing of the FMR1 gene and, consequently, the loss of the fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein that binds to G quartet forming RNA using its RGG box motif. In this study we have performed a thermodynamic analysis of the interactions between the FMRP RGG box domain and Sc1, an RNA molecule which had been previously shown to be bound with high affinity by both the full-length FMRP and by its RGG box domain. We have determined that the association between the FMRP RGG box and Sc1 RNA is dominated by hydrophobic and hydrogen bond interactions, with minor contributions from electrostatic interactions, and that the FMRP RGG box binding increases the stability of the G quartet RNA structure significantly. Interestingly, we found that the G quartet recognition is necessary but not sufficient for the FMRP RGG box binding to this RNA target, indicating that additional interactions of the peptide, possibly with the stem and/or stem-G quartet junction region, are required. Our results also indicate that the G quartet RNA recognition is not a general feature of the RGG box motif but rather carries some sequence, protein and/or RNA, specificity.

Amino Acid Sequence↗

PubMed1979

Transcription by eukaryotic RNA polymerases A and B of chromatin assembled in vitro.

Chromatin was assembled in vitro from simian virus 40 DNA form I and the calf-thymus four histones H2A, H2B, H3 and H4. Transcription with calf thymus RNA polymerases A and B (I and II) was greatly inhibited. Nucleosomes were found to inhibit both RNA chain initiation and elongation. The inhibition of elongation could be relieved by increasing ionic strength, suggesting that electrostatic interactions between histone octamer and DNA have to be broken for RNA polymerase to transcribe DNA organised into nucleosomes.

Animals↗

PubMed1999

Free energy determinants of binding the rRNA substrate and small ligands to ricin A-chain.

A continuum model is provided of the free energy terms that contribute to the molecular association of ricin A-chain (RTA) with the rRNA substrate and several small ligands. The model for RTA interactions with the RNA was taken from a previously proposed complex containing a 29-mer oligonucleotide hairpin (. Proteins 27:80-95), and models for the ligands were constructed from x-ray crystallographic structures. The calculated absolute free energies of complex formation for the RTA-RNA assembly and several single-residue substitutions are in good agreement with experimental data, given the approximations of evaluating the strain energy and conformational entropy. The free energy terms were found to resemble those of protein-protein complexes, with the net unfavorable electrostatic contribution offset by the favorable nonspecific hydrophobic effect. Decomposition of the RTA-RNA binding free energy into individual contributions revealed the electrostatic "hot" spots arising from charge-charge complementarity of the interfacial arginines with the RNA phosphate backbone. Base interactions of the GAGA loop structure dominate the hydrophobic complementarity. A linear-scaling model was parametrized for evaluating the binding of small ligands against the rRNA substrate and illustrates the free energy determinant required for designing specific RTA inhibitors.

Base Sequence↗

PubMed2001

Molecular dynamics simulation of the human U2B" protein complex with U2 snRNA hairpin IV in aqueous solution.

A 2200-ps molecular dynamics (MD) simulation of the U2 snRNA hairpin IV/U2B" complex was performed in aqueous solution using the particle mesh Ewald method to consider long-range electrostatic interactions. To investigate the interaction and recognition process between the RNA and protein, the free energy contributions resulting from individual amino acids of the protein component of the RNA/protein complex were calculated using the recently developed glycine-scanning method. The results revealed that the loop region of the U2 snRNA hairpin IV interacted mainly with three regions of the U2B" protein: 1) beta 1-helix A, 2) beta 2-beta 3, and 3) beta 4-helix C. U2 snRNA hairpin IV bound U2B" in a similar orientation as that previously described for U1 snRNA with the U1A' protein; however, the details of the interaction differed in several aspects. In particular, beta 1-helix A and beta 4-helix C in U2B" were not observed to interact with RNA in the U1A' protein complex. Most of the polar and charged residues in the interacting regions had larger mutant free energies than the nonpolar residues, indicating that electrostatic interactions were important for stabilizing the RNA/protein complex. The interaction was further stabilized by a network of hydrogen bonds and salt bridges formed between RNA and protein that was maintained throughout the MD trajectory. In addition to the direct interactions between RNA and the protein, solvent-mediated interactions also contributed significantly to complex stability. A detailed analysis of the ordered water molecules in the hydration of the RNA/protein complex revealed that bridged water molecules reside at the interface of RNA and protein as long as 2100 ps in the 2200-ps trajectory. At least 20 bridged water molecules, on average, contributed to the instantaneous stability of the RNA/protein complex. The stabilizing interaction energy due to bridging water molecules was obtained from ab initio Hartree-Fock and density functional theory calculations.

Autoantigens↗

PubMedDate not supplied

RNA solvation: a molecular dynamics simulation perspective.

With the availability of accurate methods to treat the electrostatic long-range interactions, molecular dynamics simulations have resulted in refined dynamical models of the structure of the hydration shell around RNA motifs. The models reviewed here range from basic Watson-Crick to more specific noncanonical base pairs, from "simple" double helices to RNA molecules displaying more complex tertiary folds, and from DNA/RNA hybrid double helices to RNA hybrids formed with a chemically modified strand.

Base Pairing↗

PubMed1993

RNA recognition by an isolated alpha helix.

A 17 amino acid peptide containing the arginine-rich region of the HIV Rev protein binds specifically to Rev response element (RRE) RNA. Even though it is highly charged, the peptide forms an alpha helix in solution, but only when its N- and C-termini are modified to provide favorable electrostatic interactions with the helix macrodipole. Binding affinity for IIB RNA (the primary binding site within the RRE) increases with alpha helix content, whereas nonspecific binding affinity is independent of helix content. Binding of mutant peptides demonstrates that one threonine, one asparagine, and four arginine side chains are important for sequence-specific recognition. Transactivation of the HIV LTR using Tat-Rev peptide hybrids and the RRE IIB site indicates that the peptide adopts an alpha-helical conformation in vivo. The results suggest that interactions with the RNA backbone may help to orient the alpha helix in the major groove of RNA.

Amino Acid Sequence↗

PubMed1998

Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA.

A variety of drugs inhibit biological key processes by binding to a specific RNA component. We focus here on the well-analysed hammer-head ribozyme RNA that is inhibited by aminoglycoside antibiotics, a process considered as a paradigm for studying drug/RNA interactions. With insight gained from molecular dynamics simulations of the ribozyme in the presence of Mg2+ identified by crystallography and of aminoglycosides in solution, a general model for aminoglycoside binding to RNA is proposed. A striking structurally based complementarity between the charged ammonium groups of the aminoglycosides and the metal binding sites in the hammerhead was uncovered. Despite dynamical flexibility of the aminoglycosides, several of the intramolecular distances between the charged ammonium groups of the drugs were found to be rather constant. Intramolecular ammonium distances of the aminoglycosides span ranges similar to the interionic distances between Mg2+ in the hammerhead. Successful docking of aminoglycosides to the hammerhead ribozyme could be achieved by positioning the ammonium groups at the sites occupied by Mg2+. The covalently linked ammonium groups of the aminoglycosides are thus able to complement in space the negative electrostatic potential created by a three-dimensional RNA fold. Consequently, it is suggested that aminoglycoside-derived sugars could constitute a basic set of yardstick synthons ideal for rational and combinatorial synthesis of drugs targeted at biologically relevant RNA folds.

Anti-Bacterial Agents↗

PubMed1998

Mutations in the N terminus of the brome mosaic virus polymerase affect genetic RNA-RNA recombination.

Previously, we have observed that mutations in proteins 1a and 2a, the two virally encoded components of the brome mosaic virus (BMV) replicase, can affect the frequency of recombination and the locations of RNA recombination sites (P. D. Nagy, A. Dzianott, P. Ahlquist, and J. J. Bujarski, J. Virol. 69:2547-2556, 1995; M. Figlerowicz, P. D. Nagy, and J. J. Bujarski, Proc. Natl. Acad. Sci. USA 94:2073-2078, 1997). Also, it was found before that the N-terminal domain of 2a, the putative RNA polymerase protein, participates in the interactions between 1a and 2a (C. C. Kao, R. Quadt, R. P. Hershberger, and P. Ahlquist, J. Virol. 66:6322-6329, 1992; E. O'Reilly, J. Paul, and C. C. Kao, J. Virol. 71:7526-7532, 1997). In this work, we examine how mutations within the N terminus of 2a influence RNA recombination in BMV. Because of the likely electrostatic character of 1a-2a interactions, five 2a mutants, MF1 to MF5, were generated by replacing clusters of acidic amino acids with their neutral counterparts. MF2 and MF5 retained nearly wild-type levels of 1a-2a interaction and were infectious in Chenopodium quinoa. However, compared to that in wild-type virus, the frequency of nonhomologous recombination in both MF2 and MF5 was markedly decreased. Only in MF2 was the frequency of homologous recombination reduced and the occurrence of imprecise homologous recombination increased. In MF5 there was also a 3' shift in the positions of homologous crossovers. The observed effects of MF2 and MF5 reveal that the 2a N-terminal domain participates in different ways in homologous and in nonhomologous BMV RNA recombination. This work maps specific locations within the N terminus involved in 1a-2a interaction and in recombination and further suggests that the mechanisms of the two types of crossovers in BMV are different.

Amino Acid Sequence↗

PubMed2005

Structural basis of 3' end RNA recognition and exoribonucleolytic cleavage by an exosome RNase PH core.

The exosome is a macromolecular complex that plays fundamental roles in the biogenesis and turnover of a large number of RNA species. Here we report the crystal structures of the Rrp41-Rrp42 core complex of the S. solfataricus exosome bound to short single-stranded RNAs and to ADP. The RNA binding cleft recognizes four nucleotides in a sequence-unspecific manner, mainly by electrostatic interactions with the phosphate groups. Interactions at the 2' hydroxyls of the sugars provide specificity for RNA over DNA. The structures show both the bound substrate and the cleaved product of the reaction, suggesting a catalytic mechanism for the 3'-5' phosphorolytic activity of the exosome.

Archaeal Proteins↗

PubMed2005

Kinetic analysis of the role of the tyrosine 13, phenylalanine 56 and glutamine 54 network in the U1A/U1 hairpin II interaction.

The A protein of the U1 small nuclear ribonucleoprotein particle, interacting with its stem-loop RNA target (U1hpII), is frequently used as a paradigm for RNA binding by recognition motif domains (RRMs). U1A/U1hpII complex formation has been proposed to consist of at least two steps: electrostatically mediated alignment of both molecules followed by locking into place, based on the establishment of close-range interactions. The sequence of events between alignment and locking remains obscure. Here we examine the roles of three critical residues, Tyr13, Phe56 and Gln54, in complex formation and stability using Biacore. Our mutational and kinetic data suggest that Tyr13 plays a more important role than Phe56 in complex formation. Mutational analysis of Gln54, combined with molecular dynamics studies, points to Arg52 as another key residue in association. Based on our data and previous structural and modeling studies, we propose that electrostatic alignment of the molecules is followed by hydrogen bond formation between the RNA and Arg52, and the sequential establishment of interactions with loop bases (including Tyr13). A quadruple stack, sandwiching two bases between Phe56 and Asp92, would occur last and coincide with the rearrangement of a C-terminal helix that partially occludes the RRM surface in the free protein.

Amino Acid Sequence↗

PubMed1998

Exploration of metal ion binding sites in RNA folds by Brownian-dynamics simulations.

BACKGROUND: Metal ions participate in the three-dimensional folding of RNA and provide active centers in catalytic RNA molecules. The positions of metal ions are known for a few RNA structures determined by X-ray crystallography. In addition to the crystallographically identified sites, solution studies point to many more metal ion binding sites around structured RNAs. Metal ions are also present in RNA structures determined by nuclear magnetic resonance (NMR) spectroscopy, but the positions of the ions are usually not revealed. RESULTS: A novel method for predicting metal ion binding sites in RNA folds has been successfully applied to a number of different RNA structures. The method is based on Brownian-dynamics simulations of cations diffusing under the influence of random Brownian motion within the electrostatic field generated by the static three-dimensional fold of an RNA molecule. In test runs, the crystallographic positions of Mg2+ ions were reproduced with deviations between 0.3 and 2.7 A for several RNA molecules for which X-ray structures are available. In addition to the crystallographically identified metal ions, more binding sites for cations were revealed: for example, tRNAs were shown to bind more than ten Mg2+ ions in solution. Predictions for metal ion binding sites in four NMR structures of RNA molecules are discussed. CONCLUSIONS: The successful reproduction of experimentally observed metal ion binding sites demonstrates the efficiency of the prediction method. A promising application of the method is the prediction of cation-binding sites in RNA solution structures, determined by NMR.

Base Sequence↗

PubMed2001

Probing the kinetics of formation of the bacteriophage MS2 translational operator complex: identification of a protein conformer unable to bind RNA.

We have investigated the kinetics of complex formation between bacteriophage MS2 coat protein subunits and synthetic RNA fragments encompassing the natural translational operator site, or the consensus sequences of three distinct RNA aptamer families, which are known to bind to the same site on the protein. Reactions were assayed using stopped-flow fluorescence spectroscopy and either the intrinsic tryptophan fluorescence of the protein or the signals from RNA fragments site-specifically substituted with the fluorescent adenosine analogue 2'-deoxy, 2-aminopurine. The kinetics observed were independent of the fluorophore being monitored or its position within the complex, indicating that the data report global events occurring during complex formation. Competition assays show that the complex being formed consists of a single coat protein dimer and one RNA molecule. The binding reaction is at least biphasic. The faster phase, constituting 80-85 % of the amplitude, is a largely diffusion driven RNA-protein interaction (k1 approximately 2x10(9) M(-1) s(-1)). The salt dependence of the forward reaction and the similarities of the on-rates of lower-affinity RNA fragments are consistent with a diffusion-controlled step dominated by electrostatic steering. The slower phase is independent of reactant concentration, and appears to correspond to isomerisation of the coat protein subunit(s) prior to RNA binding (k(iso) approximately 0.23 s(-1)). Measurements with a coat protein mutant (Pro78Asn) show that this phase is not due to cis-trans isomerisation at this residue. The conformational changes in the protein ligand during formation of an RNA-protein complex might play a role in the triggering of capsid self-assembly and a model for this is discussed.

Binding, Competitive↗

PubMed2001

Cloning, overexpression, and purification of aminoglycoside antibiotic nucleotidyltransferase (2'')-Ia: conformational studies with bound substrates.

Aminoglycoside nucleotidyltransferase (2'')-Ia [ANT (2'')-Ia] was cloned from Pseudomonas aeruginosa and purified from overexpressing Escherichia coli BL21(DE3) cells. The first enzyme-bound conformation of an aminoglycoside antibiotic in the active site of an aminoglycoside nucleotidyltransferase was determined using the purified aminoglycoside nucleotidyltransferase (2' ')-Ia. The conformation of the aminoglycoside antibiotic isepamicin, a psuedo-trisaccharide, bound to aminoglycoside nucleotidyltransferase (2' ')-Ia has been determined using NMR spectroscopy. Molecular modeling, employing experimentally determined interproton distances, resulted in two different enzyme-bound conformations (conformer 1 and conformer 2) of isepamicin. Conformer 1 was by far the major conformer defined by the following average glycosidic dihedral angles: PhiBC = -65.26 +/- 1.63 degrees and PsiBC = -54.76 +/- 4.64 degrees. Conformer 1 was further subdivided into one major (conformer 1a) and two minor components (conformers 1b and 1c) based on the comparison of glycosidic dihedral angles PhiAB and PsiAB. The arrangement of substrates in the enzyme.metal-ATP.isepamicin complex was determined on the basis of the measured effect of the paramagnetic substrate analogue Cr(H2O)4ATP on the relaxation rates of substrate protons which were used to determine relative distances of isepamicin protons to the Cr3+. Both conformers of isepamicin yielded arrangements that satisfied the NOE restraints and the observed paramagnetic effects of Cr(H2O)4ATP. It has been suggested that aminoglycosides use both electrostatic interactions and hydrogen bonds in binding to RNA and that the contacts made by the A and B rings to RNA are the most important for binding [Fourmy, D., Recht, M. I., Blanchard, S. C., and Puglisi, J. D. (1996) Science 274, 1367-1371]. Comparisons based on the determined conformations of enzyme-bound aminoglycoside antibiotics also suggested that interactions of rings A and B with enzymes may be the major determinant in aminoglycoside binding to enzymes [Serpersu, E. H., Cox, J. R., DiGiammarino, E. L., Mohler, M. L., Ekman, D. R., Akal-Strader, A., and Owston, M. (2000) Cell Biochem. Biophys. (in press)]. The conformation of isepamicin bound to the aminoglycoside nucleotidyltransferase (2' ')-Ia, determined in this work, lent further support to this theory. Furthermore, comparison of enzyme-bound conformations of isepamicin to the RNA-bound conformation of gentamycin C1a also showed remarkable similarities between the enzyme-bound and RNA-bound aminoglycoside antibiotic conformations. These studies should aid in the design of effective inhibitors possessing a broad range of aminoglycoside-modifying enzymes as targets.

Bacterial Proteins↗

PubMed1979

Cation-induced regulatory mechanism of GTPase activity dependent on polypeptide initiation factor 2.

Initiation factor IF-2 ribosome dependent GTP hydrolysis (uncoupled GTPase) presents a bell-shaped pH profile which is shifted by changes in ionic strength. At low ionic strength (I = 25 mM) the maximal hydrolytic activity occurs at pH 7.5; when the ionic strength is increased the pH optimum of the reaction is shifted toward more acidic values. Such behavior can be satisfactorily explained as the effect of an electrostatic potential developed by a neighboring polyanion, presumably RNA, on the catalytic site. The addition of fMet-tRNAfMet or AcPhe-tRNAPhe and messenger RNA (coupled GTPase) changes the ionic strength--pH characteristics of the reaction. Thus there is an effect, direct or indirect, of components located at the ribosomal P site. Investigation of the effect of neighboring polyanions on the catalytic activity of the factor-dependent ribosomal GTPases can be seen to provide information about their functional significance that is complementary to that gained from direct structural studies.

Escherichia coli↗

PubMed1980

Role of interaction energy in the specificity of transcription. I-The Watson Crick G-C base pair template.

The purpose of this work is to show that the selectivity of the nucleotide bases in RNA transcription c an be inferred, in principle, from the DNA base pair - RNA base interaction. The catalytic role of enzymes in this process is, therefore, only to form the sugar - phosphate backbone. A systematic study for the evaluation of the interaction energy of the DNA base pair with the enterant RNA bases have been undertaken to elucidate the aforesaid mechanism. Electrostatic hard sphere approximation of Nash and Bradley 1 has been employed. Non bonded induced dipole and London dispersion forces are not taken into account. The present communication gives the results of computations of the interaction energy of the four RNA bases. The results have been discussed with reference to Stent's and Zubay's schemes of RNA transcription.

Base Composition↗

PubMed2005

Smart polymeric micelles as nanocarriers for oligonucleotides and siRNA delivery.

The development of in vivo delivery systems for oligonucleotides and siRNA is strongly desired to achieve their clinical applications. Recently, polyplex micelles, which are formed through an electrostatic interaction between nucleic acid compounds (DNA and RNA) and poly(ethylene glycol) (PEG)-polycation block copolymers, have received much attention due to their nanometric-scaled size and excellent biocompatibility. Here, three types of newly engineered block copolymers were developed to construct polyplex micelles useful for oligonucleotides and siRNA delivery: (1) PEG-polycation diblock copolymers possessing diamine side-chain with distinctive pKa for siRNA encapsulation into polyplex micelles with high endosomal escaping ability, (2) Lactosylated PEG-(oligonucleotide or siRNA) conjugate through acid-labile beta-thiopropionate linkage to construct pH-sensitive PIC micelles, and (3) PEG-poly(methacrylic acid) block copolymer for the construction of organic/inorganic hybrid nanoparticles encapsulating siRNA.

Hydrogen-Ion Concentration↗

PubMed1998

Solution structure of P22 transcriptional antitermination N peptide-boxB RNA complex.

We have determined the solution structure of a 15-mer boxB RNA hairpin complexed with a 20-mer basic peptide of the N protein involved in bacteriophage P22 transcriptional antitermination. Complex formation involves adaptive binding with the N peptide adopting a bent alpha-helical conformation that packs tightly through hydrophobic and electrostatic interactions against the major groove face of the boxB RNA hairpin, orienting the open opposite face for potential interactions with host factors and/or RNA polymerase. Four nucleotides in the boxB RNA hairpin pentaloop form a stable GNRA like tetraloop structural scaffold on complex formation, allowing the looped out fifth nucleotide to make extensive hydrophobic contacts with the bound peptide. The guanidinium group of a key arginine is hydrogen-bonded to the guanine in a loop-closing sheared G.A mismatch and to adjacent backbone phosphates. The identified intermolecular contacts account for the consequences of N peptide and boxB RNA mutations on bacteriophage transcriptional antitermination.

Amino Acid Sequence↗

PubMed2005

Structure of a putative 2'-5' RNA ligase from Pyrococcus horikoshii.

Cyclic phosphodiesterase and 2'-5' RNA ligase are members of a superfamily of proteins which share structural similarities even though their homology may be very low. A putative 2'-5' RNA ligase from Pyrococcus horikoshii has been crystallized and its X-ray crystallographic structure determined to 2.4 A. The protein crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.07, b = 45.47, c = 93.17 A and one protein monomer in the asymmetric unit. The molecular-replacement probe was a 2'-5' RNA ligase from Thermus thermophilus which shares 30% sequence identity. The P. horikoshii RNA ligase has some structural features that have more in common with a cyclic phosphodiesterase from Arabidopsis thaliana with which it has no significant homology, yet an examination of the electrostatic surface potential clearly defines its relationship to the T. thermophilus RNA ligase. However, the size of the active-site cleft is smaller and less positively charged than that of the T. thermophilus homologue, suggesting that the actual substrate may be smaller than that previously postulated for the latter.

Binding Sites↗