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PubMed1982

Binding of spermidine to transfer ribonucleic acid.

The binding of spermidine to yeast tRNAPhe and Escherichia coli tRNAGlu2 at low and high ionic strength was studied by equilibrium dialysis. Once corrected for the expected Donnan effect, the binding at low ionic strength obeys the simple relationship of equivalent binding sites, and cooperative binding of spermidine to tRNA could not be detected. At low ionic strength (0.013 M Na+ ion), tRNAPhe (yeast) has 13.9 +/- 2.3 strong spermidine binding sites per molecule with Kd = 1.39 X 10(-6) M and a few weak spermidine binding sites which were inaccessible to experimentation; tRNAGlu2 (E. coli) has 14.8 +/- 1.6 strong spermidine binding sites and 4.0 +/- 0.1 weak spermidine binding sites with Kd = 1.4 X 10(-6) M and Kd = 1.23 X 10(-4) M, respectively. At high ionic strength (0.12 M monovalent cation) and 0.01 M Mg2+, tRNAPhe (yeast) has approximately 13 strong spermidine binding sites with an apparent Kd = 3.4 X 10(-3) M while the dimeric complex tRNAPhe X tRNAGlu2 has 10.4 +/- 1.2 strong spermidine binding sites per monomer with an apparent Kd = 2.0 X 10(-3) M. In the presence of increasing Na+ ion or K+ ion concentration, spermidine binding data do not fit a model for competitive binding to tRNA by monovalent cations. Rather, analysis of binding data by the Debye-Hückel approximation results in a good fit of experimental data, indicating that monovalent cations form a counterion atmosphere about tRNA, thus decreasing electrostatic interactions. On the basis of equilibrium binding analyses, it is proposed that the binding of spermidine to tRNA occurs predominantly by electrostatic forces.

Mathematics↗

PubMed1993

Theoretical analyses on the role of metal cations in RNA cleavage processes.

Previously, we found that locations of metal cations in biomolecules, such as proteins and nucleotides, were in accord with predictions based on electrostatic-potential calculations of the nearest polar functional groups. Electrostatic potential of a cyclic oxyphosphorane dianion 2, which is a model compound for an intermediate or a transition state for the hammerhead ribozyme reaction, indicates that Mg2+ coordinations at the regions between axial (bridging) and equatorial (phosphoryl) oxygens are most favorable. If we consider the fact that negative charge is concentrated on two of the equatorial phosphoryl oxygens in the transition state 2, the axial-equatorial coordination is an unexpected result. Nevertheless, experimental results also support such an axial-equatorial coordination. Therefore, hammerhead ribozyme is a metalloenzyme in which at least one Mg2+ ion acts as a base and possibly a second Mg2+ ion acts as a Lewis acid. The double Mg2+ ion catalysis may be a common mechanism among various types of ribozyme and some protein enzymes as well.

Animals↗

PubMed2006

A novel stable RNA pentaloop that interacts specifically with a motif peptide of lambda-N protein.

To achieve a novel specific peptide-nucleic acid binding model, we designed an in vitro selection procedure to decrease the energetic contribution of the electrostatic interaction in the total binding energy and to increase the contribution of hydrogen bonding and pi-pi stacking. After the selection of hairpin-loop RNAs that specifically bound to a model peptide of lambda N protein (N peptide), a new thermostable pentaloop RNA motif (N binding thermostable RNA hairpin: NTS RNA) was revealed. The obtained NTS RNA was able to bind to the N peptide with superior specificity to the boxB RNA, which is the naturally occurring partner of the lambda N protein.

Amino Acid Motifs↗

PubMed2003

YY1-DNA interaction results in a significant change of electronic context as measured by capacitance.

The detailed mechanism behind the processes of DNA-dependent RNA transcription initiation is largely unknown. When transcription initiation factors bind DNA, a significant change in the electrostatic state of the complex can result. Using electrical capacitance measurements of solutions of the YY1 zinc finger transcription initiation factor and the adeno-associated viral P5 promoter DNA, we observed a specific dielectric change when a protein-DNA complex was formed. We propose that complexation results in electrostatic changes that may trigger the markedly different electrical behavior, and offer a possible explanation for our results.

Adenoviridae↗

PubMed2005

Insights into SARS-CoV transcription and replication from the structure of the nsp7-nsp8 hexadecamer.

Coronavirus replication and transcription machinery involves multiple virus-encoded nonstructural proteins (nsp). We report the crystal structure of the hexadecameric nsp7-nsp8 supercomplex from the severe acute respiratory syndrome coronavirus at 2.4-angstroms resolution. nsp8 has a novel 'golf-club' fold with two conformations. The supercomplex is a unique hollow, cylinder-like structure assembled from eight copies of nsp8 and held tightly together by eight copies of nsp7. With an internal diameter of approximately 30 angstroms, the central channel has dimensions and positive electrostatic properties favorable for nucleic acid binding, implying that its role is to confer processivity on RNA-dependent RNA polymerase.

Amino Acid Sequence↗

PubMed2006

Calculation of pKas in RNA: on the structural origins and functional roles of protonated nucleotides.

pK(a) calculations based on the Poisson-Boltzmann equation have been widely used to study proteins and, more recently, DNA. However, much less attention has been paid to the calculation of pK(a) shifts in RNA. There is accumulating evidence that protonated nucleotides can stabilize RNA structure and participate in enzyme catalysis within ribozymes. Here, we calculate the pK(a) shifts of nucleotides in RNA structures using numerical solutions to the Poisson-Boltzmann equation. We find that significant shifts are predicted for several nucleotides in two catalytic RNAs, the hairpin ribozyme and the hepatitis delta virus ribozyme, and that the shifts are likely to be related to their functions. We explore how different structural environments shift the pK(a)s of nucleotides from their solution values. RNA structures appear to use two basic strategies to shift pK(a)s: (a) the formation of compact structural motifs with structurally-conserved, electrostatic interactions; and (b) the arrangement of the phosphodiester backbone to focus negative electrostatic potential in specific regions.

Catalysis↗

PubMed2002

The linkage between magnesium binding and RNA folding.

Understanding the linkage between Mg(2+) binding and RNA folding requires a proper theoretical model describing the energetics of Mg(2+) binding to the folded and unfolded states of RNA. Our current understanding of Mg(2+) binding to these different RNA states derives from empirical thermodynamic models that depend on a number of unjustified assumptions. We present a rigorous theoretical model describing the linkage between RNA folding and magnesium ion binding. In this model, based on the non-linear Poisson-Boltzmann (NLPB) equation, the stabilization of RNA by Mg(2+) arises from two distinct binding modes, diffuse binding and site binding. Diffusely bound Mg(2+) are described as an ensemble of hydrated ions that are attracted to the negative charge of the RNA. Site-bound Mg(2+) are partially desolvated ions that are attracted to electronegative pockets on the RNA surface. We explore two systems, yeast tRNA(Phe) and a 58-nucleotide rRNA fragment, with different Mg(2+) binding properties. The NLPB equation accurately describes both the stoichiometric and energetic linkage between Mg(2+) binding and RNA folding for both of these systems without requiring any fitted parameters in the calculation. Moreover, the NLPB model presents a well-defined physical description of how Mg(2+) binding helps fold an RNA. For both of the molecules studied here, the relevant unfolded state is a disordered intermediate state (I) that contains stable helical secondary structure without any tertiary contacts. Diffusely bound Mg(2+) interact with these secondary structure elements to stabilize the I state. The secondary structural elements of the I state fold into a compact, native tertiary structure (the N state). Diffuse binding plays a dominant role in stabilizing the N state for both RNAs studied. However, for the rRNA fragment, site-binding to a location with extraordinarily high electrostatic potential is also coupled to folding. Our results suggest that much experimental data measuring the linkage between Mg(2+) binding and RNA folding must be reinterpreted.

Escherichia coli↗

PubMed2006

Prediction of molecular alignment of nucleic acids in aligned media.

We demonstrate--using the data base of all deposited DNA and RNA structures aligned in Pf1-medium and RDC refined--that for nucleic acids in a Pf1-medium the electrostatic alignment tensor can be predicted reliably and accurately via a simple and fast calculation based on the gyration tensor spanned out by the phosphodiester atoms. The rhombicity is well predicted over its full range from 0 to 0.66, while the alignment tensor orientation is predicted correctly for rhombicities up to ca. 0.4, for larger rhombicities it appears to deviate somewhat more than expected based on structural noise and measurement error. This simple analytical approach is based on the Debye-Huckel approximation for the electrostatic interaction potential, valid at distances sufficiently far away from a poly-ionic charged surface, a condition naturally enforced when the charge of alignment medium and solute are of equal sign, as for nucleic acids in a Pf1-phage medium. For the usual salt strengths and nucleic acid sizes, the Debye-Huckel screening length is smaller than the nucleic acid size, but large enough for the collective of Debye-Huckel spheres to encompass the whole molecule. The molecular alignment is then purely electrostatic, but it's functional form is under these conditions similar to that for steric alignment. The proposed analytical expression allows for very fast calculation of the alignment tensor and hence RDCs from the conformation of the nucleic acid molecule. This information provides opportunities for improved structure determination of nucleic acids, including better assessment of dynamics in (multi-domain) nucleic acids and the possibility to incorporate alignment tensor prediction from shape directly into the structure calculation process. The procedures are incorporated into MATLAB scripts, which are available on request.

Bacteriophage Pf1↗

PubMed1980

Distances between 3' ends of ribosomal ribonucleic acids reassembled into Escherichia coli ribosomes.

The three ribonucleic acids (RNAs) from Escherichia coli ribosomes were isolated and then labeled at their 3' ends by oxidation with periodate followed by reaction with thiosemicarbazides of fluorescein or eosin. Ribosomal subunits reconstituted with the labeled RNAs were active for polyphenylalanine synthesis. The distances between the 3' ends of the RNAs in 70S ribosomes were estimated by nonradiative energy transfer from fluorescein to eosin. The percentage of energy transfer was calculated from the decrease in fluorescence lifetime of fluorescein in the quenched sample compared to the unquenched sample. Fluorescence lifetime was measured in real time by using a mode-locked laser for excitation and a high-speed electrostatic photomultiplier tube for detection of fluorescence. The distances between fluorophores attached to the 3' ends of 16S RNA and 5S RNA or 23S RNA were estimated to be about 55 and 71 A, respectively. The corresponding distance between the 5S RNA and 23S RNA was too large to be measured reliably with the available probes but was estimated to be greater than 65 A. Comparison of the quantum yields of the labeled RNAs free in solution and reconstituted into ribosomal subunits suggests that the 3' end of 16S RNA does not interact appreciably with other ribosomal components and may be in a relatively exposed position, whereas the 3' ends of the 5S RNA and 23S RNA may be buried in the 70S ribosomal subunit.

Chemical Phenomena↗

PubMed1995

Interaction of intermediate filaments with ribosomes in vitro.

Cytoplasmic intermediate filaments (cIFs) should be capable of loosely interacting with negatively charged, macromolecular assemblies, given that the net positive charge of the N-terminal head domains exposed on the surface of the cIFs is not fully neutralized by the neighboring C-terminal tail domains and the filament body proper. In order to substantiate this notion, cIFs reconstituted in vitro from various type III IF proteins and obtained as constituents of whole cell mount preparations were allowed to react at physiological ionic strength with 40S and 60S ribosomal subunits as well as 80S run-off ribosomes of mammalian origin. Electron micrographs of the reaction products show colocalization of all kinds of ribosomal particles with both the reconstituted and the natural cIFs, including the cytokeratin filaments of whole cell mount preparations of epithelial cells. The ribosome-cIF complexes are sensitive to ionic strength higher than physiological as well as to mild treatment with pancreatic RNase A, suggesting the direct involvement of the RNA moieties of the ribosomal particles in the binding reaction via the engagement of ionic bonds. This weak, electrostatic interaction potential of the cIF complement of the cytoskeleton might be exploited by the eukaryotic cell for the storage and distribution in the cytoplasm of non-polyribosomal ribonucleoprotein particles of the protein-synthesizing machinery and possibly also of negatively charged membrane vesicles.

Animals↗

PubMed2004

Crystal structure of RumA, an iron-sulfur cluster containing E. coli ribosomal RNA 5-methyluridine methyltransferase.

RumA catalyzes transfer of a methyl group from S-adenosylmethionine (SAM) specifically to uridine 1939 of 23S ribosomal RNA in Escherichia coli to yield 5-methyluridine. We determined the crystal structure of RumA at 1.95 A resolution. The protein is organized into three structural domains: The N-terminal domain contains sequence homology to the conserved TRAM motif and displays a five-stranded beta barrel architecture characteristic of an oligosaccharide/oligonucleotide binding fold. The central domain contains a [Fe(4)S(4)] cluster coordinated by four conserved cysteine residues. The C-terminal domain displays the typical SAM-dependent methyltransferase fold. The catalytic nucleophile Cys389 lies in a motif different from that in DNA 5-methylcytosine methyltransferases. The electrostatic potential surface reveals a predominately positively charged area that covers the concave surface of the first two domains and suggests an RNA binding mode. The iron-sulfur cluster may be involved in the correct folding of the protein or may have a role in RNA binding.

Amino Acid Motifs↗

PubMed2005

Conformational dynamics of RNA-peptide binding: a molecular dynamics simulation study.

Molecular dynamics simulations of the binding of the heterochiral tripeptide KkN to the transactivation responsive (TAR) RNA of HIV-1 is presented, using an all-atom force field with explicit water. To obtain starting structures for the TAR-KkN complex, semirigid docking calculations were performed that employ an NMR structure of free TAR RNA. The molecular dynamics simulations show that the starting structures in which KkN binds to the major groove of TAR (as it is the case for the Tat-TAR complex of HIV-1) are unstable. On the other hand, the minor-groove starting structures are found to lead to several binding modes, which are stabilized by a complex interplay of stacking, hydrogen bonding, and electrostatic interactions. Although the ligand does not occupy the binding position of Tat protein, it is shown to hinder the interhelical motion of free TAR RNA. The latter is presumably necessary to achieve the conformational change of TAR RNA to bind Tat protein. Considering the time evolution of the trajectories, the binding process is found to be ligand-induced and cooperative. That is, the conformational rearrangement only occurs in the presence of the ligand and the concerted motion of the ligand and a large part of the RNA binding site is necessary to achieve the final low-energy binding state.

Binding Sites↗

PubMedDate not supplied

[Interaction of the antineoplastic agent mitoxantrone with double-stranded nucleic acids].

The binding of mitoxantrone with double-helical nucleic acids was investigated by the methods of isothermal microcalorimetry, circular dichroism and absorption at the ionic strength mu = 0.11 and 0.011 M NaCl at temperature region of 30 divided by 60 degrees C. The investigation shows, that at mu = 0.11 M NaCl mitoxantrone interacts with double-helical nucleic acids in one way only. For such conditions using spectrophotometric titration data Scatchard plots for the binding of mitoxantrone with double-helical nucleic acids were constructed. The calculations show that the saturation stoichiometry is one mitoxantrone molecule per 2 divided by 3 base pairs DNA and 6 divided by 8 base pairs RNA. The dependence of binding constant from GC-content is observed. It is shown that the binding enthalpy of mitoxantrone with DNA and RNA increases linearly and reaches -(3.0 +/- 0.5) kkal per 1 mol mitoxantrone. It is shown that a binding mitoxantrone with double-helical nucleic acids, besides the intercalation of rings, a determinate contribution in the binding is given also by electrostatic interaction of side chains mitoxantrone with nucleic acids.

Animals↗

PubMed2005

Crystal structure of a ribonuclease P protein Ph1601p from Pyrococcus horikoshii OT3: an archaeal homologue of human nuclear ribonuclease P protein Rpp21.

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the removal of 5' leader sequences from tRNA precursors (pre-tRNA). The human protein Rpp21 is essential for human RNase P activity in tRNA processing in vitro. The crystal structure of Ph1601p from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, the archaeal homologue of Rpp21, was determined using the multiple anomalous dispersion (MAD) method with the aid of anomalous scattering in zinc and selenium at 1.6 A resolution. Ph1601p comprises an N-terminal domain (residues 1-55), a central linker domain (residues 56-79), and a C-terminal domain (residues 80-120), forming an L-shaped structure. The N-terminal domain consists of two long alpha-helices, while the central and C-terminal domains fold in a zinc ribbon domain. The electrostatic potential representation indicates the presence of positively charged clusters along the L arms, suggesting a possible role in RNA binding. A single zinc ion binds the well-ordered binding site that consists of four Cys residues (Cys68, Cys71, Cys97, and Cys100) and appears to stabilize the relative positions of the N- and C-domains. Mutations of Cys68 and Cys71 or Cys97 and Cys100 to Ser destabilize the protein structure, which results in inactivation of the RNase P activity. In addition, site-directed mutagenesis suggests that Lys69 at the central loop and Arg86 and Arg105 at the zinc ribbon domain are strongly involved in the functional activity, while Arg22, Tyr44, Arg65, and Arg84 play a modest role in the activity.

Amino Acid Sequence↗

PubMed2005

A uniform mechanism correlating dangling-end stabilization and stacking geometry.

The geometry of the dangling base in 105 published structures (from X-ray/NMR) containing single-stranded overhangs has been analyzed and correlated to the thermodynamic stabilization found (UV) for the corresponding dangling base/closing basepair combination in short oligonucleotides. The study considers most combinations of closing basepairs, sequence and dangling base residue type, attached in both the 3'- and 5'-ends of both DNA and RNA. Linear regression analysis showed a straightforward correlation (R = 0.873) between the degree of screening for the hydrogen bonds of the closing basepair provided by the dangling base and the resulting thermodynamic stabilization in both DNA and RNA series with dangling ends either at the 3'- or at the 5'-terminus. Regression analysis of only the datasets from RNA gives an improved correlation, R = 0.934, showing that dangling ends on RNA are more ordered than the dangling ends on DNA, R = 0.376. This study highlights the gain in the free energy of stabilization owing to the favorable stacking between the dangling nucleobase and the neighboring basepair and the resulting strengthening of the hydrogen bond of the closing basepair. By acting as a hydrophobic cap on the terminal of the DNA or RNA duplex, the dangling-end residue restricts the bulk water access to the terminal basepair, thereby providing it with a microenvironment devoid of water, which consequently enhances its thermodynamic stability, making it energetically comparable to the corresponding internal basepair. Thus, one single structural model consisting of the interplay of the above electrostatic interactions can be used to explain the molecular basis of the observed thermodynamic effects for dangling-end attachment to the 3'- and 5'-ends of both DNA and RNA duplexes, which is a key step toward accurate dangling-end effect prediction.

Nucleic Acid Conformation↗

PubMed1987

Mechanism of DNA (Southern) and protein (Western) blotting on cellulose nitrate and other membranes.

The transfer of DNA fractions from hydrophilic gels to nitrocellulose membranes (Southern blotting) which was soon followed by the description of an analogous procedure for RNA (Northern blotting), and somewhat later for proteins (Western blotting), has rapidly become an important separation and characterization method in molecular biology, genetic engineering, and immunological detection. Surface tension measurements have shown that the interfacial attraction between DNA and cellulose esters (-delta G132) in aqueous media can be considerable. The weaker binding energy of proteins to cellulose nitrate and to cellulose acetate may be compared to hydrophobic interaction chromatography, as on account of the somewhat lower [-delta G132] values, it often is necessary to "fix" them more tightly onto nitrocellulose by using high salt concentrations. The binding energy of RNA to both cellulose esters also is rather low. In addition to the effect of high ionic strength, the effect of adding methanol, and the effects of denaturation, heating and drying on the energy of attachment of the biopolymers to cellulose esters, have been studied. Cationized nylon membranes have been advocated recently, especially for electrophoretic transfer of nucleic acids (in which process high salt concentrations cannot easily be used). With positively charged nylon membranes, the attachment mainly occurs through the electrostatic attraction between the strongly negatively charged nucleic acids (or proteins) and the positively charged membrane. Also, more apolar membranes (of polyvinyl difluoride) have been proposed, which manifest a strong interfacial (hydrophobic) attraction to all the above biopolymers (regardless of their electrostatic charge). However, with these two novel membrane types it is no longer possible to exploit the large difference in binding energy between DNA and RNA, which makes cellulose nitrate membranes so uniquely suited for RNA-DNA hybridization assays.

Animals↗

PubMed1996

Calculations of nucleic acid conformations.

The present computational power and sophistication of theoretical approaches to nucleic acid structural investigation are sufficient for the realization of static and dynamic models that correlate accurately with current crystallographic, NMR and solution-probing structural data, and consequently are able to provide valuable insights and predictions for a variety of nucleic acid conformational families. In molecular dynamics simulations, the year 1995 was marked by the foray of fast Ewald methods, an accomplishment resulting from several years' work in the search for an adequate treatment of the electrostatic long-range forces so primordial in nucleic acid behavior. In very large systems, and particularly in the RNA-folding field, techniques originating from artificial intelligence research, like constraint satisfaction programming or genetic algorithms, have established their utility and potential.

DNA↗

PubMed2000

Replacement of the phosphorodiester linkages of RNA with guanidinium linkages: the solid-phase synthesis of ribonucleic guanidine.

[reaction: see text] Replacement of the negatively charged phosphodiester linkages of RNA with positively charged guanidinium linkages provides the polycationic ribonucleic guanidine (RNG). RNG is anticipated to bind strongly to target DNA/RNA through the specific interactions of nucleobases and the attractive electrostatic interactions of backbones. Preparation of building blocks and the solid-phase synthesis of RNG are reported. Both trimeric and pentameric uridyl RNG have been synthesized.

Guanidines↗