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At least 91 records · Page 5Linked to original sources
PubMed1996

The affinity of an oligodeoxynucleotide-peptide conjugate for an RNA hairpin loop depends on stereochemistry at the DNA-peptide junction.

Molecular models of an oligodeoxynucleotide-peptide conjugate complexed to an RNA hairpin loop were constructed to assess the effect of stereoisomerism at the point of attachment of the peptide to the oligodeoxynucleotide on the affinity of the conjugate for an RNA target. The peptide portion of the oligodeoxynucleotide-peptide conjugate, (L-lysine)8, was covalently attached to the N-allyl group of (D)- or (L)-aspartic alcohol that was incorporated into the interior of an antisense oligodeoxynucleotide. The stereocenter in the oligodeoxynucleotide interior originates from either (D)- or (L)-aspartic alcohol. The oligodeoxynucleotide portion of the oligodeoxynucleotide-peptide conjugate forms Watson-Crick base pairs with the single-stranded RNA that flanks the RNA hairpin loop. The positively charged peptide makes specific electrostatic contacts with the negatively charged phosphate backbone of the RNA hairpin loop when attached to the N-allyl of (D)-aspartic alcohol but does not have the proper orientation to make these electrostatic contacts when attached to the N-allyl of (L)-aspartic alcohol. This modelling study emphasizes the importance of stereocontrol at the point of branching in synthesizing oligodeoxynucleotide-peptide conjugates for binding of RNA hairpin loops.

Aspartic Acid↗
PubMed2003

A thermodynamic framework for the magnesium-dependent folding of RNA.

The goal of this review is to present a unified picture of the relationship between ion binding and RNA folding based on recent theoretical and computational advances. In particular, we present a model describing how the association of magnesium ions is coupled to the tertiary structure folding of several well-characterized RNA molecules. This model is developed in terms of the nonlinear Poisson-Boltzmann (NLPB) equation, which provides a rigorous electrostatic description of the interaction between Mg(2+) and specific RNA structures. In our description, most of the ions surrounding an RNA behave as a thermally fluctuating ensemble distributed according to a Boltzmann weighted average of the mean electrostatic potential around the RNA. In some cases, however, individual ions near the RNA may shed some of their surrounding waters to optimize their Coulombic interactions with the negatively charged ligands on the RNA. These chelated ions are energetically distinct from the surrounding ensemble and must be treated explicitly. This model is used to explore several different RNA systems that interact differently with Mg(2+). In each case, the NLPB equation accurately describes the stoichiometric and energetic linkage between Mg(2+) binding and RNA folding without requiring any fitted parameters in the calculation. Based on this model, we present a physical description of how Mg(2+) binds and stabilizes specific RNA structures to promote the folding reaction.

Binding Sites↗
PubMed2006

Specific RNA binding to ordered phospholipid bilayers.

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA-membrane interactions.

Binding Sites↗
PubMed2002

Biologically important reactions catalyzed by RNA molecules.

The last few years have seen a considerable increase in our understanding of catalysis by naturally occurring RNA molecules called ribozymes. The biological functions of RNA molecules depend upon their adoption of appropriate three-dimensional structures. The structure of RNA has a very important electrostatic component, which results from the presence of charged phosphodiester bonds. Metal ions are usually required to stabilize the folded structures and/or catalysis. Some ribozymes utilize metal ions as catalysts, whereas others use the ions to maintain appropriate three-dimensional structures. In the latter case, the correct folding of the RNA structures can perturb the pKa values of the nucleotide(s) within a catalytic pocket such that they act as general acid/bases catalysts.

Base Sequence↗
PubMed1999

Hydrophilic and cationic latex particles for the specific extraction of nucleic acids.

The adsorption of BSA and RNA onto hydrophilic and thermosensitive poly(N-isopropyl-acrylamide) (NIPAM) latex particles was described as a function of pH, ionic strength and temperature. The hydrogel poly(NIPAM) latex was synthesized by precipitation polymerization in the presence of a cationic amino-containing monomer. The latex obtained was characterized in terms of particle size, and electrophoretic mobility as a function of pertinent variables: pH, temperature and ionic strength. The adsorption of BSA onto the latex was investigated to identify the conditions at which the adsorbed amount of BSA was negligible. The adsorption of RNA was studied to establish the conditions which give rise to maximal adsorption of RNA. In order to favor the desorption of RNA, desorption was investigated by changing the pH, ionic strength, and temperature. The adsorption of BSA was found to be lower at 20 than at 40 degrees C. However, the adsorption of RNA is drastically affected by the pH and the ionic strength of the medium. Maximal adsorbed amounts were obtained at acidic pH, 20 degrees C, and low ionic strength. The adsorption is shown to decrease when the pH, temperature and ionic strength increase, implying that the adsorption was mainly governed by electrostatic interactions. Maximal release of RNA molecules was obtained at high ionic strength and basic pH.

Adsorption↗
PubMed2003

Crystal structure of NusA from Thermotoga maritima and functional implication of the N-terminal domain.

We report the crystal structure of N-utilizing substance A protein (NusA) from Thermotoga maritima (TmNusA), a protein involved in transcriptional pausing, termination, and antitermination. TmNusA has an elongated rod-shaped structure consisting of an N-terminal domain (NTD, residues 1-132) and three RNA binding domains (RBD). The NTD consists of two subdomains, the globular head and the helical body domains, that comprise a unique three-dimensional structure that may be important for interacting with RNA polymerase. The globular head domain possesses a high content of negatively charged residues that may interact with the positively charged flaplike domain of RNA polymerase. The helical body domain is composed of a three-helix bundle that forms a hydrophobic core with the aid of two neighboring beta-strands. This domain shows structural similarity with one of the helical domains of sigma(70) factor from Escherichia coli. One side of the molecular surface shows positive electrostatic potential suitable for nonspecific RNA interaction. The RBD is composed of one S1 domain and two K-homology (KH) domains forming an elongated RNA binding surface. Structural comparison between TmNusA and Mycobacterium tuberculosis NusA reveals a possible hinge motion between NTD and RBD. In addition, a functional implication of the NTD in its interaction with RNA polymerase is discussed.

Amino Acid Sequence↗
PubMed1998

Deciphering RNA recognition: aminoglycoside binding to the hammerhead ribozyme.

Aminoglycoside antibiotics inhibit protein biosynthesis and various ribozymes. Structural electrostatic complementarity can explain the inhibition mechanism of the hammerhead ribozyme: positively charged ammonium groups match the negatively charged metal-ion-binding pockets created by the RNA fold's electrostatic field.

Aminoglycosides↗
PubMed2000

Spectinomycin inhibits the self-splicing of the group 1 intron RNA.

Effects of the aminoglycoside spectinomycin on the self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td) have been investigated. The kinetic analysis demonstrated that spectinomycin acts as a mixed noncompetitive inhibitor for the td intron RNA with a K(i) of 7.2 mM. Increasing the spectinomycin concentration raised the K(m) values with the corresponding decrease of V(max) and k(cat) values. The specificity of the splicing inhibition by spectinomycin is due to changes in both K(m) and k(cat). The splicing inhibition by spectinomycin is dependent on pH changes and Mg(2+) concentration, indicating electrostatic interactions with the intron RNA. It has been proposed that the key structural features in spectinomycin responsible for the inhibition of splicing may be the hydroxyl groups on the antibiotic.

Guanosine Triphosphate↗
PubMed2002

Mechanism of the gBP21-mediated RNA/RNA annealing reaction: matchmaking and charge reduction.

The guide RNA-binding protein gBP21 has been characterized as a mitochondrial RNA/RNA annealing factor. The protein co-immunoprecipitates with RNA editing ribonucleoprotein complexes, which suggests that gBP21 contributes its annealing activity to the RNA editing machinery. In support of this view, gBP21 was found to accelerate the hybridization of cognate guide (g)RNA/pre-edited mRNA pairs. Here we analyze the mechanism of the gBP21-mediated RNA annealing reaction. Three possible modes of action are considered: chaperone function, matchmaker function and product stabilization. We conclude that gBP21 works as a matchmaker by binding to gRNAs as one of the two RNA annealing reactants. Three lines of evidence substantiate this model. First, gBP21 and gRNAs form a thermodynamically and kinetically stable complex in a 1 + 1 stoichiometry. Secondly, gRNA-bound gBP21 stabilizes single-stranded RNA, which can be considered the transition state in the annealing reaction. Thirdly, gBP21 has a low affinity for double-stranded RNAs, suggesting the release of the annealed reaction product after the hybridization step. In the process, up to six ionic bonds are formed between gBP21 and a gRNA, which decreases the net negative charge of the RNA. As a consequence, the electrostatic repulsion between the two annealing reactants is reduced favoring the hybridization reaction.

Adsorption↗
PubMed1994

Structural features of adenovirus 2 virus-associated RNA required for binding to the protein kinase DAI.

The double-stranded RNA activated protein kinase DAI contains an RNA binding domain consisting of two copies of a double-stranded RNA binding motif. We have investigated the role of RNA structure in the interaction between DAI and the structured single-stranded RNA, adenovirus VA RNAI, which inhibits DAI activation. Mutations in the apical stem, terminal stem, and central domain of the RNA were tested to assess the contribution of these elements to DAI binding in vitro. The data demonstrate that over half a turn of intact apical stem is required for the interaction and that there is a correlation between the binding of apical stem mutants and their ability to function both in vivo and in vitro. There was also evidence of preference for GC-rich sequence in the proximal region of the apical stem. In the central domain the correlation between binding and function of mutant RNAs was poor, suggesting that at least some of this region plays no direct role in binding to DAI, despite its functional importance. Exceptionally, central domain mutations that encroached on the phylogenetically conserved stem 4 of VA RNA disrupted binding, and complementary mutations in this sequence partially restored binding. Measurement of the binding of wild-type VA RNAI to DAI and p20, a truncated form of the protein containing the RNA binding domains alone, under various ionic conditions imply that the major interactions are electrostatic and occur via the protein's RNA binding domain. However, differences between full-length DAI and p20 in their binding to mutants in the conserved stem suggest that regions outside the RNA binding domain also participate in the binding. The additional interactions are likely to be non-ionic, and may be important for preventing DAI activation during virus infection.

Adenoviruses, Human↗
PubMed2003

Insights into the regioselectivity and RNA-binding affinity of HIV-1 nucleocapsid protein from linear-scaling quantum methods.

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) plays several important roles in the viral life-cycle and presents an attractive target for rational drug design. Here, the macromolecular reactivity of NC and its binding to RNA is characterized through determination of electrostatic and chemical descriptors derived from linear-scaling quantum calculations in solution. The computational results offer a rationale for the experimentally observed susceptibility of the Cys49 thiolate toward small-molecule electrophilic agents, and support the recently proposed stepwise protonation mechanism of the C-terminal Zn-coordination complex. The distinctive binding mode of NC to SL2 and SL3 stem-loops of the HIV-1 genomic RNA packaging signal is studied on the basis of protein side-chain contributions to the electrostatic binding energies. These results indicate the importance of several basic residues in the 3(10) helical region and the N-terminal zinc finger, and rationalize the presence of several evolutionarily conserved residues in NC. The combined reactivity and RNA-binding study provides new insights that may contribute toward the structure-based design of anti-HIV therapies.

Cysteine↗
PubMed2001

A thermodynamic framework for Mg2+ binding to RNA.

We present a model describing how Mg(2+) binds and stabilizes specific RNA structures. In this model, RNA stabilization arises from two energetically distinct modes of Mg(2+) binding: diffuse- and site-binding. Diffusely bound Mg(2+) are electrostatically attracted to the strong anionic field around the RNA and are accurately described by the Poisson-Boltzmann equation as an ensemble distributed according to the electrostatic potentials around the nucleic acid. Site-bound Mg(2+) are strongly attracted to specifically arranged electronegative ligands that desolvate the ion and the RNA binding site. Thus, site-binding is a competition between the strong coulombic attraction and the large cost of desolvating the ion and its binding pocket. By using this framework, we analyze three systems where a single site-bound Mg(2+) may be important for stability: the P5 helix and the P5b stem loop from the P4-P6 domain of the Tetrahymena thermophila group I intron and a 58-nt fragment of the Escherichia coli 23S ribosomal RNA. Diffusely bound Mg(2+) play a dominant role in stabilizing these RNA structures. These ions stabilize the folded structures, in part, by accumulating in regions of high negative electrostatic potential. These regions of Mg(2+) localization correspond to ions that are observed in the x-ray crystallographic and NMR structures of the RNA. In contrast, the contribution of site-binding to RNA stability is often quite small because of the large desolvation penalty. However, in special cases, site-binding of partially dehydrated Mg(2+) to locations with extraordinarily high electrostatic potential can also help stabilize folded RNA structures.

Magnesium↗
PubMed2004

Crystal structure of KD93, a novel protein expressed in human hematopoietic stem/progenitor cells.

The crystal structure of a novel hypothetical protein, KD93, expressed in human hematopoietic stem/progenitor cells, was determined at 1.9A resolution using the multiple-wavelength anomalous dispersion (MAD) method. The protein KD93, which is encoded by the open reading frame HSPC031, is a NIP7 homologue and belongs to the UPF0113 family. The structural and functional information for the group of homologues has not yet been determined. Crystallographic analysis revealed that the overall fold of KD93 consists of two interlinked alpha/beta domains. Structure-based homology analysis with DALI revealed that the C domain of KD93 matches the PUA domain of some RNA modification enzymes, especially that of archaeosine tRNA-ribosyltransferase (ArcTGT), which suggests that its possible molecular function is related to RNA binding. The difference between the RNA binding regions of KD93 and ArcTGT in amino acid constitution and surface electrostatic potential indicate that they may have different RNA binding modes. The N domain of KD93 is a unique structure with no obvious similarity to other proteins with known three-dimensional structures. The high-resolution structure of KD93 provides a first view of a member of the family of hypothetical proteins. And the structure provides a framework to deduce and assay the molecular function of other proteins of the UPF0113 family.

Crystallography, X-Ray↗
PubMed2005

Chemiluminescence-based electrophoretic mobility shift assay of RNA-protein interactions: application to binding of viral capsid proteins to RNA.

A chemiluminescence electrophoretic mobility shift assay was introduced for the study of RNA-protein interactions that include association of genomic RNA and viral capsid proteins. Binding of the capsid protein of Venezuelan equine encephalitis virus (VEEV) to several types of RNA was used as a model system to test the application of the method. The effects of RNA secondary structures and the significance of electrostatic interaction on binding were identified. This method may have wide application to the study of RNA-protein interactions.

Capsid Proteins↗
PubMed2006

Electrostatic properties of cowpea chlorotic mottle virus and cucumber mosaic virus capsids.

Electrostatic properties of cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CMV) were investigated using numerical solutions to the Poisson-Boltzmann equation. Experimentally, it has been shown that CCMV particles swell in the absence of divalent cations when the pH is raised from 5 to 7. CMV, although structurally homologous, does not undergo this transition. An analysis of the calculated electrostatic potential confirms that a strong electrostatic repulsion at the calcium-binding sites in the CCMV capsid is most likely the driving force for the capsid swelling process during the release of calcium. The binding interaction between the encapsulated genome material (RNA) inside of the capsid and the inner capsid shell is weakened during the swelling transition. This probably aids in the RNA release process, but it is unlikely that the RNA is released through capsid openings due to unfavorable electrostatic interaction between the RNA and capsid inner shell residues at these openings. Calculations of the calcium binding energies show that Ca(2+) can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca(2+) ions can induce the capsid contraction and stabilize the native form.

Binding Sites↗
PubMed2005

QSAR study for mycobacterial promoters with low sequence homology.

The general belief is that quantitative structure-activity relationship (QSAR) techniques work only for small molecules and, protein sequences or, more recently, DNA sequences. However, with non-branched graph for proteins and DNA sequences the QSAR often have to be based on powerful non-linear techniques such as support vector machines. In our opinion, linear QSAR models based on RNA could be useful to assign biological activity when alignment techniques fail due to low sequence homology. The idea bases the high level of branching for the RNA graph. This work introduces the so-called Markov electrostatic potentials (k)xi(M) as a new class of RNA 2D-structure descriptors. Subsequently, we validate these molecular descriptors solving a QSAR classification problem for mycobacterial promoter sequences (mps), which constitute a very low sequence homology problem. The model developed (mps=-4.664.(0)xi(M)+0. 991.(1)xi(M)-2.432) was intended to predict whether a naturally occurring sequence is an mps or not on the basis of the calculated (k)xi(M) value for the corresponding RNA secondary structure. The RNA-QSAR approach recognises 115/135mps (85.2%) and 100% of control sequences. Average predictability and robustness were greater than 95%. A previous non-linear model predicts mps with a slightly higher accuracy (97%) but uses a very large parameter space for DNA sequences. Conversely, the (k)xi(M)-based RNA-QSAR encodes more structural information and needs only two variables.

Bacterial Proteins↗
PubMed1999

Protonated 2'-aminoguanosine as a probe of the electrostatic environment of the active site of the Tetrahymena group I ribozyme.

We have probed the electrostatic environment of the active site of the Tetrahymena group I ribozyme (E) using protonated 2'-aminoguanosine (), in which the 2'-OH of the guanosine nucleophile (G) is replaced by an group. At low concentrations of divalent metal ion (2 mM Mg(2+)), binds at least 200-fold stronger than G or G(NH)()2, with a dissociation constant of </=1 microM from the ribozyme. oligonucleotide substrate. complex (). This strong binding suggests that the group interacts with negatively charged phosphoryl groups within the active site. Increasing the concentration of divalent metal ion weakens the binding of to E. S more than 10(2)-fold. The Mn(2+) concentration dependence suggests that M(C), the metal ion that interacts with the 2'-moiety of G in the normal reaction, is responsible for this effect. M(C) and compete for binding to the active site; this competition could arise from electrostatic repulsion between the positively charged and M(C) and, possibly, from their competition for interaction with active site phosphoryl groups. The reactive phosphoryl group of S increases the competition between M(C) and, consistent with a network of interactions involving M(C) that help position the reactive phosphoryl group and the guanosine nucleophile with respect to one another. The chemical step with bound is at least 10(4)-fold slower than with G or G(NH)()2. These results provide additional support for an integral role of M(C) in catalysis by the Tetrahymena ribozyme, and demonstrate the utility of the moiety as an electrostatic probe within a structured RNA.

Amines↗