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PubMed1993

RNA-binding patterns in total human tissue proteins: analysis by northwestern blotting.

We have developed a reproducible Northwestern (NW) blotting method to identify general patterns of RNA-binding proteins in total human tissue homogenates and have identified some of the factors contributing to this reproducibility. Unfractionated homogenates of human brain tissue were separated by SDS-PAGE, electrotransferred wet to nitrocellulose, and probed with in-vitro-transcribed labeled RNAs. Approximately ten size classes of RNA-binding proteins were observed consistently and reproducibly. Although sequence-independent electrostatic RNA-protein interactions likely contributed to most of the binding, binding to some proteins was shown to be more dependent on protein conformation: binding was not blocked by preincubation with single- or double-stranded DNA, nor with poly(A) RNA, but preincubation with tRNA revealed a distinct subset of RNA-binding proteins. In addition, preincubation with RNA, but not DNA, revealed a previously undetected RNA-binding protein of approximately 90 kDa. The NW blotting method described here can be used to reveal tissue-specific differences in RNA-binding patterns.

Amyloid beta-Protein Precursor↗
PubMed2006

Electrostatic properties of promoter recognized by E. coli RNA polymerase Esigma70.

A comparative analysis of electrostatic patterns for 359 sigma70-specific promoters and 359 nonpromoter regions on electrostatic map of Escherichia coli genome was carried out. It was found that DNA is not a uniformly charged molecule. There are some local inhomogeneities in its electrostatic profile which correlate with promoter sequences. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which differ for different promoter groups and may be involved as signal elements in differential recognition of various promoters by the enzyme. Some specific electrostatic elements which are responsible for modulating promoter activities due to ADP-ribosylation of RNA polymerase alpha-subunit were found in far upstream regions of T4 phage early promoters and E. coli ribosomal promoters.

Base Sequence↗
PubMed2002

A compact RNA tertiary structure contains a buried backbone-K+ complex.

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate groups of a hairpin loop within a large tertiary core. During refinement of an X-ray crystal structure containing this RNA, a potassium ion was found to be contacted by six oxygen atoms from the buried phosphate groups; the ion is contained completely within the solvent-accessible surface of the RNA. The electrostatic potential at the ion chelation site is unusually large, and more than compensates for the substantial energetic penalties associated with partial dehydration of the ion and displacement of delocalized ions. The very large predicted binding free energy, approximately -30 kcal/mol, implies that the site must be occupied for the RNA to fold. These findings agree with previous studies of the ion-dependent folding of tertiary structure in this RNA, which concluded that a monovalent ion was bound in a partially dehydrated environment where Mg2+ could not easily compete for binding. By compensating the unfavorable free energy of buried phosphate groups with a chelated ion, the RNA is able to create a larger and more complex tertiary fold than would be possible otherwise.

Base Sequence↗
PubMed1976

RNA viruses: stabilization of brome mosaic virus.

The relative importances of protein-protein and RNA-protein interactions in stabilizing the architecture of brome mosaic virus particles are discussed in the light of the following experimental evidence: (a) disassembly pathways of the virus particles, (b) reassembly of the virus and self-association capacity of the protein moiety, and (c) the role of divalent cations in virus stabilization, and their relevance to localization of the RNA in the virus particles. Evidence is given that the capsid of BMV is primarily stabilized by hydrophobic bonds at low pH, but not around and above neutrality where RNA-protein electrostatic interactions are essential to the integrity of the virus particles. A model is proposed for the structure of BMV in the different configurational states.

Capsid↗
PubMed1988

Comparative calorimetric studies on the dynamic conformation of plant 5S rRNA. I. Thermal unfolding pattern of lupin seeds and wheat germ 5S rRNAs, also in the presence of magnesium and sperminium cations.

An attempt has been made to correlate differential scanning calorimetry melting profiles of 5S rRNAs from lupin seeds (L.s.) and wheat germ (W.g.) with their structure. It is suggested that the observed differences in thermal unfolding are due to differences in RNA nucleotide sequence and as a consequence in higher order structures. Interesting effects induced by magnesium cation, perprotonated and permethylated sperminium tetracations are discussed. It is suggested that the difference in the stabilizing effect of the three cations results from different mode of their interactions with RNA. "Pure" electrostatic interactions expected for permethylated tetracations are rather weak due to the steric hindrance around each positively charged nitrogen atom. Electrostatic interactions of the other two cations are significantly enhanced by coordination bonding for magnesium and by hydrogen bonding for protonated sperminium cation.

Calorimetry, Differential Scanning↗
PubMed1999

Docking of cationic antibiotics to negatively charged pockets in RNA folds.

The binding of aminoglycosides to RNA provides a paradigm system for the analysis of RNA-drug interactions. The electrostatic field around three-dimensional RNA folds creates localized and defined negatively charged regions which are potential docking sites for the cationic ammonium groups of aminoglycosides. To explore in RNA folds the electronegative pockets suitable for aminoglycoside binding, we used calculations of the electrostatic field and Brownian dynamics simulations of cation diffusion. We applied the technique on those RNA molecules experimentally known to bind aminoglycosides, namely, two tobramycin aptamers (Wang, Y.; Rando, R. R. Chem. Biol. 1995, 2, 281-290): the aminoglycoside-binding region in 16S ribosomal RNA (Moazed, S.; Noller, H. F. Nature 1987, 327, 389-394) and the TAR RNA from human immunodeficiency virus (Mei, H.-Y.; et al. Bioorg. Med. Chem. Lett. 1995, 5, 2755-2760). For the aptamers and ribosomal RNA, for which the binding sites of the aminoglycosides are known, a good agreement between negatively charged pockets and the binding positions of the drugs was found. On the basis of variations between neomycin-like and kanamycin-like aminoglycosides in the interaction with the electrostatic field of ribosomal RNA, we propose a model for the different binding specificities of these two classes of drugs. The spatial congruence between the electronegative pockets in RNA folds and binding positions of aminoglycosides was used to dock aminoglycosides to ribosomal and TAR RNAs. Molecular dynamics simulations were used to analyze possible RNA-drug interactions. Aminoglycosides inhibit the binding of the viral Tat protein to TAR RNA; however, the drug-binding sites are still unknown. Thus, our docking approach provides first structural models for TAR-aminoglycoside complexes. The RNA-drug interactions observed in the modeled complexes support the view that the antibiotics might lock TAR in a conformation with low affinity for the Tat protein, explaining the experimentally found aminoglycoside inhibition of the Tat-TAR interaction (Mei, H.-Y.; et al. Bioorg. Med. Chem. Lett. 1995, 5, 2755-2760).

Anti-Bacterial Agents↗
PubMed2000

RNA polymerase--promoter recognition. Specific features of electrostatic potential of "early" T4 phage DNA promoters.

Comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken, along with calculation of topography of electrostatic potential around the native and ADP-ribosylated C-terminal domain of RNA polymerase alpha-subunit. The data obtained indicate that there is specific difference in the patterns of electrostatic potential distribution in far upstream regions of T4 promoters differing by their response to ADP-ribosylation of RNA polymerase. A specific change in profiles of electrostatic potential distribution for the native and ADP-ribosylated forms of RNA polymerase alpha-subunit was observed suggesting that this factor may be responsible for modulating T4 promoter activities in response to the enzyme modification.

Adenosine Diphosphate Ribose↗
PubMed2000

Recognition of a cognate RNA aptamer by neomycin B: quantitative evaluation of hydrogen bonding and electrostatic interactions.

Aminoglycosides are an important class of antibiotic that selectively target RNA structural motifs. Recently we have demonstrated copper derivatives of amino-glycosides to be efficient cleavage agents for cognate RNA motifs. To fully develop their potential as pharmaceutical agents it is necessary to understand both the structural mechanisms used by aminoglycosides to target RNA, and the relative contributions of hydrogen bonding and electrostatic interactions to recognition selectivity. Herein we report results from a calorimetric analysis of a stem-loop 23mer RNA aptamer complexed to the aminoglycoside neomycin B. Key thermodynamic parameters for complex formation have been determined by isothermal titration calorimetry, and from the metal-ion dependence of these binding parameters the relative contributions of electrostatics and hydrogen bonding toward binding affinity have been assessed. The principal mechanism for recognition and binding of neomycin B to the RNA major groove is mediated by hydrogen bonding.

Base Sequence↗
PubMed2007

Folding cooperativity in RNA and DNA is dependent on position in the helix.

Secondary structural motifs play essential roles in the folding and function of RNA and DNA molecules. Previous work from our lab compared the folding of small DNA and RNA hairpin loops containing a sheared GA pair [Moody, E. M., Feerar, J. C., and Bevilacqua, P. C. (2004) Biochemistry 43, 7992-7998]. We found that the small DNA hairpins fold in a highly cooperative manner with indirect coupling, while their RNA counterparts fold in a much less cooperative fashion and display direct coupling. Herein, we extend this study to the double-stranded helix. We carried out double mutant cycles on base pairs having identical nearest-neighbor contexts but located in either external or internal helical registers. In the external register, both RNA and DNA exhibit extensive folding cooperativity between the penultimate and terminal base pair, which is independent of mismatch identity. In contrast, DNA exhibits virtually no folding cooperativity in the center of the helix, while RNA maintains substantial coupling, which is dependent on mismatch identity. Two models account for these non-nearest-neighbor effects: one involves the unfavorable entropy of helix initiation common to DNA and RNA, and the other involves steric and electrostatic strain peculiar to RNA. These data show that RNA can display cooperativity less than, greater than, or equal to that of DNA depending on context and position.

Base Pair Mismatch↗
PubMed1996

RNA hairpins with non-nucleotide spacers bind efficiently to the human U1A protein.

The N-terminal RNA binding domain (RBD1) of the human U1A protein binds to the ten nucleotide loop in stemloop II of of U1 snRNA, and to its own 3' UTR structure. The nucleotides critical for recognition by the U1A RBD are displayed in very different geometric contexts in these two targets, leading to the question of what common features of RNA structure allow the RBD to efficiently recognize these two RNAs. The experiments described here used RNA hairpins, in which the loop size was altered by deletion, insertion or substitution with non-nucleotide (ethylene glycol)n spacers, to determine what features of this RNA structure were critical for interaction with the RBD1. Substitution of the three nucleotides on the 3' side of the RNA hairpin loop by (ethylene glycol)6-18 spacers does not significantly perturb the affinity, energetics or electrostatics of this RNA: protein association. These results confirm the suggestion that these loop nucleotides provide a flexible tether to allow the other seven nucleotides to fit onto the binding surface of the RBD, and lead to the hypothesis that conformational flexibility and the possible end-to-end distance of seven loop nucleotides are critical features of this complex formation.

Base Sequence↗
PubMed2026

RNA-repelling Anionic Clusters in Human Rhinovirus Cooperate with Cationic Residues to Promote Virion Assembly and Restrain RNA Release.

Research on virus nucleic acid-protein interactions is important to understand infection and guide antiviral drug design. In previous studies we showed that the human rhinovirus (RV) genomic RNA is organized as a dodecahedral cage formed by 30 RNA duplex elements anchored to capsid concavities. We showed also that capsid-RNA duplex interactions include conserved tryptophans, neutral polar residues, and many positively charged residues that promote virion assembly and restrain RNA release by stabilizing the negatively charged RNA duplex structure. The present study expands our understanding of the capsid-RNA duplex interface in RV by addressing the structural and functional roles of conserved patches of negatively charged capsid residues interposed between each RNA duplex and its binding site at the capsid inner surface. The initial hypothesis was that electrostatic repulsion between anionic residues and RNA phosphates would lead to functional effects opposite to those previously found for cationic residues that can electrostatically attract RNA phosphates. In fact, those anionic residues do not oppose, but act together with cationic residues at the RNA duplex binding sites to promote virion assembly and restrain RNA release. Cryogenic electron microscopy analysis showed that negatively charged residues at the capsid-RNA duplex interfaces have a different structural role than positively charged residues, even though they all play similar functional roles. A tentative model is discussed to explain the functional effects of the complex distribution of negative and positive electrostatic potential found at capsid-RNA duplex interfaces in RV.

capsid↗
PubMed2004

Recognition of planar and nonplanar ligands in the malachite green-RNA aptamer complex.

Ribonucleic acids are an attractive drug target owing to their central role in many pathological processes. Notwithstanding this potential, RNA has only rarely been successfully targeted with novel drugs. The difficulty of targeting RNA is at least in part due to the unusual mode of binding found in most small-molecule-RNA complexes: the ligand binding pocket of the RNA is largely unstructured in the absence of ligand and forms a defined structure only with the ligand acting as scaffold for folding. Moreover, electrostatic interactions between RNA and ligand can also induce significant changes in the ligand structure due to the polyanionic nature of the RNA. Aptamers are ideal model systems to study these kinds of interactions owing to their small size and the ease with which they can be evolved to recognize a large variety of different ligands. Here we present the solution structure of an RNA aptamer that binds triphenyl dyes in complex with malachite green and compare it with a previously determined crystal structure of a complex formed with tetramethylrosamine. The structures illustrate how the same RNA binding pocket can adapt to accommodate both planar and nonplanar ligands. Binding studies with single- and double-substitution mutant aptamers are used to correlate three-dimensional structure with complex stability. The two RNA-ligand complex structures allow a discussion of structural changes that have been observed in the ligand in the context of the overall complex structure. Base pairing and stacking interactions within the RNA fold the phosphate backbone into a structure that results in an asymmetric charge distribution within the binding pocket that forces the ligand to adapt through a redistribution of the positive partial charge.

Coloring Agents↗
PubMed2003

Tandem electrostatic effect from the first to the third aglycon in the trimeric RNA owing to the nearest-neighbor interaction.

We here show an electrostatic polar-pi interaction from the first to the third aglycon, via the second aglycon, in the ground state in two single stranded trimeric RNAs, 5'-GpA(1)pA(2)-3' (3) and 5'-GpApC-3' (4), as a result of intramolecular nearest neighbor offset-stacking. The experimental evidence in support of this conclusion has been obtained by comparing the pK(a)s of each aglycone in the two trimers with those of guanosine 3'-ethyl phosphate, GpEt (1) and 5'-GpA-3' (2): Thus, the pK(a) of N(1)-H of guanin-9-yl of 5'-GpA(1)pA(2)-3' (3) could be measured by pH titration (pH 7.3-11.6) of its own deltaH8G (pK(a) 9.75 +/- 0.02) as well as from deltaH8A(1) (pK(a) 9.72 +/- 0.02) and deltaH2A(1) (pK(a) 9.83 +/- 0.04) of the neighboring pA(1)p moiety and the deltaH8A(2) (pK(a) 9.83 +/- 0.02) of the terminal pA(2) moiety. Similarly, the pH titration of GpApC (4) shows the pK(a) of N(1)-H of guanin-9-yl from its own deltaH8G (pK(a) 9.88 +/- 0.03) as well as from deltaH8A (pK(a) 9.87 +/- 0.01) of the neighboring pAp moiety, and deltaH5/H6C (pK(a) 9.88 +/- 0.01 and 9.90 +/- 0.01 respectively) of the 3'-terminal cytosin-1-yl. This intramolecular nearest neighbor electrostatic interaction in the single-stranded RNA modulates the pseudoaromaticity of the nearest neighbors by almost total transmission of because they constitute an extended array of offset-stacked coupled aromatic heterocycles within a polyanionic sugar-phosphate backbone at the ground state. The enhanced basicity of Gp residue by ca. 0.6 pK(a) unit in the trimers compared to that of the dimer is a result of the change in the electrostatic microenvironment owing to the nearest neighbors in the former (the nucleobases as well as the phosphates). Thus, the from the 5'-guanylate ion to the 3'-end aglycon via the central adenin-9-yl is 55 to 56 kJ mol(-)(1) in each step through a distance spanning approximately 6.8 A in an unfolded state. As a result, the pK(a) of guanin-9-yl moiety has become 9.25 +/- 0.02 in GpEt (1), 9.17 +/- 0.02 in GpA (2), 9.75 +/- 0.02 in GpApA (3), and 9.88 +/- 0.03 in GpApC (4). This means that guanin-9-yl moiety of trimers 3 and 4 is more basic than in the monomer or the dimer. The net outcome of this electrostatic cross-talk between the two neighboring heterocycles is creation of new hybrid aglycones in an oligo or polynucleotide, whose physicochemical property and the pseudoaromatic character are completely dependent both upon the nearest neighbors, and whether they are stacked or unstacked. Thus, this tunable physicochemical property of an aglycon (an array of the extended genetic code) may have considerable implication in our understanding of the specific ligand binding ability of an aptamer, the pK(a) and the hydrogen bonding ability of nucleic acids in a microenvironment, or in the triplet usage by the anticodon-codon interaction in the protein biosynthesis in the ribosome.

Hydrogen-Ion Concentration↗
PubMed2005

Recognition of the spliceosomal branch site RNA helix on the basis of surface and electrostatic features.

We have investigated electrostatic and surface features of an essential region of the catalytic core of the spliceosome, the eukaryotic precursor messenger (pre-m)RNA splicing apparatus. The nucleophile for the first of two splicing reactions is the 2'-hydroxyl (OH) of the ribose of a specific adenosine within the intron. During assembly of the spliceosome's catalytic core, this adenosine is positioned by pairing with a short region of the U2 small nuclear (sn)RNA to form the pre-mRNA branch site helix. The solution structure of the spliceosomal pre-mRNA branch site [Newby,M.I. and Greenbaum,N.L. (2002) Nature Struct. Biol., 9, 958-965] showed that a phylogenetically conserved pseudouridine (psi) residue in the segment of U2 snRNA that pairs with the intron induces a markedly different structure compared with that of its unmodified counterpart. In order to achieve a more detailed understanding of the factors that contribute to recognition of the spliceosome's branch site helix and activation of the nucleophile for the first step of pre-mRNA splicing, we have calculated surface areas and electrostatic potentials of psi-modified and unmodified branch site duplexes. There was no significant difference between the total accessible area or ratio of total polar:nonpolar groups between modified and unmodified duplexes. However, there was substantially greater exposure of nonpolar area of the adenine base, and less exposure of the 2'-OH, in the psi-modified structure. Electrostatic potentials computed using a hybrid boundary element and finite difference nonlinear Poisson-Boltzmann approach [Boschitsch, A.H. and Fenley, M.O. (2004) J. Comput. Chem., 25, 935-955] revealed a region of exceptionally negative potential in the major groove surrounding the 2'-OH of the branch site adenosine. These surface and electrostatic features may contribute to the overall recognition of the pre-mRNA branch site region by other components of the splicing reaction.

Adenosine↗
PubMed1997

Emerging themes in RNA folding.

RNAs, like proteins, readily form specific structures adapted for ligand binding and catalysis. Since they are composed of completely different chemical building blocks, however, RNAs and proteins necessarily use distinct strategies to assemble complex architectures. While burial of hydrophobic residues drives protein folding, the hydrophobic effect in RNA contributes primarily to the formation of secondary structure. To form tertiary structure, RNA must overcome electrostatic repulsions from the phosphate backbone. How do negatively charged double helices pack together to produce catalytic centers and ligand binding surfaces? Here, we review our understanding of the principles that underlie RNA folding based on the structural information currently available.

Base Sequence↗
PubMed2006

Cooperative binding and self-assembling behavior of cationic low molecular-weight dendrons with RNA molecules.

Tri(ethylene glycol) derived, low molecular-weight dendrons with various amine end groups were synthesized and characterized for their properties of binding and self-assembling with RNA using the Candida ribozyme as a model RNA molecule. These dendritic compounds form stable complexes and well-defined nanoscale particles with RNA molecules via electrostatic interactions and self-assembly process, while leaving the other terminal of the tri(ethylene glycol) chain accessible for targeting. This suggests that dendrimers of this type hold great promise for specific RNA targeting and RNA delivery.

Candida↗
PubMed1998

Molecular dynamics investigations of hammerhead ribozyme RNA.

The hammerhead ribozyme, a small catalytic RNA molecule, cleaves, in the presence of magnesium ions, a specific phosphodiester bond within its own backbone, leading to 23-cyclic phosphate and 5-OH extremities. In order to study the dynamical flexibility of the hammerhead RNA, we performed molecular dynamics simulations of the solvated crystal structure of an active hammerhead ribozyme, obtained after flash-freezing crystals soaked with magnesium. Because of a careful equilibration protocol and the use of the Ewald summation in calculating the electrostatic interactions, the RNA structure remained close to the crystal structure, as attested by a root-mean-square deviation below 2.5 A after 750 ps of simulation. All Watson-Crick base pairs were intact at the end of the simulations. The tertiary interactions, such as the sheared G.A pairs and the U-turn, important for the stabilisation of the three-dimensional RNA fold, were also retained. The results demonstrate that molecular dynamics simulations can be successfully used to investigate the dynamical behaviour of a ribozyme, thus, opening a road to study the role of transient structural changes involved in ribozyme catalysis.

Base Pairing↗