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PubMed1996

Residues in the RNP1-like sequence motif of Rho protein are involved in RNA-binding affinity and discrimination.

The termination of transcription in Escherichia coli by action of Rho factor is dependent on the ability of this homohexameric protein to make productive interactions with the nascent RNA molecule to be terminated. The roles of two residues in a phylogenetically conserved sequence motif in the RNA-binding domain of Rho, Asp60 and Phe62, were analyzed by studies of the biochemical properties of pure mutant proteins. F62S Rho had greatly reduced affinity for lambda cro RNA, very poor ability to terminate transcription in vitro by itself and only partial termination activity (at a level consistent with its in vivo defect) in the presence of NusG. D60G Rho had a high affinity for lambda cro RNA but a much lower ability to discriminate against RNA molecules lacking cis-acting Rho-utilization sequences, and a reduced efficiency of termination that was not improved by NusG. These results indicate a major role for Phe62 in stabilizing the binding of Rho to RNA through hydrophobic interactions, while Asp60 provides an electrostatic repulsive force that allows a rapid dissociation of non-productive complexes with RNA.

Adenosine Triphosphatases↗

PubMed1993

Dissection of the his leader pause site by base substitution reveals a multipartite signal that includes a pause RNA hairpin.

A key feature of transcriptional attenuation in some amino acid biosynthetic operons is a transcriptional pause that occurs immediately after synthesis of the first leader transcript secondary structure. Both RNA secondary structure and downstream DNA sequence are important for pausing at these sites; however, the precise RNA structures involved and the relative contribution of other RNA and DNA bases to pausing are unknown. We studied the effects of base substitutions upstream from the his leader pause site (immediately prior to addition of G103) to determine how nucleic acid sequences and RNA structure contribute to pausing. By testing compensatory base substitutions, we found that pausing depended in part on an RNA secondary structure containing a five base-pair stem and eight nucleotide loop, which we call the his pause RNA hairpin. The his pause hairpin forms 11 nucleotides upstream from the paused transcript 3' end and thus corresponds to only the upper portion of the larger his A:B leader transcript secondary structure. Some base substitutions in the ten nucleotides between the pause hairpin and the 3' end of the transcript increased pausing, whereas others decreased pausing. However, compensatory substitutions that restored pairing of these bases in the lower portion of the A:B secondary structure did not alter these effects. Changing the 3'-terminal nucleotide of the transcript (U102) altered both the position and strength of pausing. Thus, in addition to the downstream DNA sequence, three distinct segments of nucleic acid upstream from the nucleotide-addition site in the transcription complex contribute to pausing in different ways: the pause RNA hairpin, the 3'-proximal region of transcript or DNA template, and the 3'-terminal nucleotide. We suggest that electrostatic interaction between the pause hairpin and RNA polymerase, rather than disruption of an RNA:DNA heteroduplex, delays elongation at the his leader pause site.

Bacterial Proteins↗

PubMed1994

Costabilization of peptide and RNA structure in an HIV Rev peptide-RRE complex.

An arginine-rich peptide corresponding to amino acids 34-50 of the human immunodeficiency virus Rev protein has been shown to bind specifically to its RNA-binding site (RRE) when the peptide is in an alpha-helical conformation. Mutation of any one of six amino acids (Thr34, Arg35, Arg38, Arg39, Asn40, or Arg44) was shown to strongly decrease specific RNA-binding affinity in vitro, suggesting that these residues may contact specific bases or distinct structural features of the RNA. We now show that the four arginine side chains, and not just their charge, are important for specific binding in vivo, and present evidence that three additional arginines (Arg46, Arg48, and Arg50) may make electrostatic contacts to the RRE. RNA-binding specificity of the Rev peptide is temperature-dependent in vitro, correlating with alpha-helix unfolding. Circular dichroism experiments indicate that the peptide helical structure is stabilized when bound specifically to the RRE and that the RNA undergoes a conformational change upon binding. Because the structures of the peptide and RNA in this model system appear to be mutually stabilized upon binding, it is suggested that the entire complex may be viewed as a single folding unit.

Amino Acid Sequence↗

PubMed1975

Evidence for the presence in calf thymus of a peptidic factor controlling DNA transcription in vitro.

A thymic factor causes a strong inhibition of the DNA-directed RNA polymerase reaction in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (less than 5000). The biological activity of the thymic factor cannot be attributed to the presence of a nuclease or of a histone fragment. The RNA synthesis is controlled by this factor by means of electrostatic interactions between the peptide compound and DNA. Inhibitory activity on RNA synthesis was absent from kidney extracts.

Animals↗

PubMed2006

Aptamer to ribozyme: the intrinsic catalytic potential of a small RNA.

The discovery of RNA-based catalysis 23 years ago dramatically changed the way biologists and biochemists thought of RNA. In the recent past, several ribozymes structures have provided some answers as to how catalysis is accomplished and how it relates to RNA structure and folding. However, there is still little information as to how catalytic activity evolved. Here we show that the small malachite green-binding aptamer has intrinsic catalytic potential that can be realized by designing the proper substrate. The charge distribution within the RNA binding pocket stabilizes the transition state of an ester hydrolysis reaction and thus accelerates the overall reaction. The results suggest that electrostatic forces can contribute significantly to RNA-based catalysis. Moreover, even simple RNA structures that have not been selected for catalytic properties can have a basic catalytic potential if they encounter the right substrate. This provides a possible starting point for the molecular evolution of more complex ribozymes.

Aptamers, Nucleotide↗

PubMed2003

Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen.

The N terminal region of hepatitis delta antigen (HDAg), referred to here as NdAg, has a nucleic acid chaperone activity that modulates the ribozyme activity of hepatitis delta virus (HDV) RNA and stimulates hammerhead ribozyme catalysis. We characterized the nucleic acid binding properties of NdAg, identified the structural and sequence domains important for nucleic acid binding, and studied the correlation between the nucleic acid binding ability and the nucleic acid chaperone activity. NdAg does not recognize the catalytic core of HDV ribozyme specifically. Instead, NdAg interacts with a variety of nucleic acids and has higher affinities to longer nucleic acids. The studies with RNA homopolymers reveal that the binding site size of NdAg is around nine nucleotides long. The extreme N terminal portion of NdAg, the following coiled-coil domain and the basic amino acid clusters in these regions are important for nucleic acid binding. The nucleic acid-NdAg complex is stabilized largely by electrostatic interactions. The formation of RNA-protein complex appears to be a prerequisite for facilitating hammerhead ribozyme catalysis of NdAg and its derivatives. Mutations that reduce the RNA binding activity or high ionic strength that destabilizes the RNA-protein complex, reduce the nucleic acid chaperone activity of NdAg.

Amino Acid Sequence↗

PubMed1998

Ion-RNA interactions in the RNA pseudoknot of a ribosomal frameshifting site: molecular modeling studies.

The three-dimensional (3-D) structure of a RNA pseudoknot that causes the efficient ribosomal frameshifting in the gag-pro region of mouse mammary tumor virus (MMTV) has been determined recently by nuclear magnetic resonance (NMR) studies. But since the structure refinement in the studies did not use metal ions and waters, it is not clear how metal ions participate in the stabilization of the pseudoknot, and what kind of ion-RNA interactions dominate in the tertiary contacts for the RNA pseudoknotting. Based on the reported structure data of the pseudoknot VPK of MMTV, we gradually refined the structure by restrained molecular dynamics (MD) using NMR distance restraints. Restrained MD simulation of the RNA pseudoknot was performed with sodium ions and water molecules. Our results are in good agreement with known NMR data and delineate the importance of the metal ion coordination in the stability of the pseudoknot. In the non-coaxially stacking pseudoknot, stem 1 (S1), stem 2 (S2), and the intervening A14 involves unconventional stacking of base pairs coordinated by Na+ and/or bridging water molecules. A6 and G7 of loop L1 make a perfect base stacking in the major groove and are further stabilized by coordinated Na+ ions and water molecules. The first 4-nucleotide (nt) ACUC of loop L2 form a sharp turn and the following 4-nt AAAA cross the minor groove of S1 and are steadied by interactions with the nucleotides of S , bridging water molecules and coordinated Na+ ions. Our studies suggest that the metal ion plays a crucial role in the RNA pseudoknotting of VPK. In the stacking interior of S1 and S2, the Na+ ion is positioned in the major groove and interacts directly with the carbonyl group O6 of G28 and carbonyl group O4 of U13 in the wobble base pair U13:G28. The ion-RNA interactions in MMTV VPK not only stabilize the RNA pseudoknot but also modify the electrostatic properties of the nucleotides at the critical parts of the pseudoknot VPK.

Animals↗

PubMed2004

Characterization of tectoRNA assembly with cationic conjugated polymers.

Association between RNAs with preprogrammed molecular recognition units can be quantified by using cationic, water-soluble conjugated polymers. The method uses a fluorophore-labeled probe RNA (RNA-F*), which is treated with a target structure (RNA-T). Heterodimer formation, (RNA-T/RNA-F*), increases the total negative charge on the F*-bearing macromolecule and reduces the number of negatively charged molecules (relative to unbound RNA-T+ RNA-F*). On the basis of electrostatic interactions, we anticipated more effective binding between CCP and (RNAT/RNA-F*), a reduction of the average CCP- - -F* distance, and more effective FRET upon excitation of the conjugated polymer. The resulting signals benefit from the optical amplification characteristic of emissive conjugated polymers. Solution dissociation constants can be determined by monitoring F* intensity changes as a function of [RNA-F*] and the ratio: [I(T) - I(NB)]/I(NB), where I(T) and I(NB) are the F* intensities in the presence of the target RNA (RNA-T) and a nonbinding RNA (RNA-NB), respectively, while keeping the concentration of the conjugated polymer constant. By focusing on [I(T) - I(NB)]/I(NB) as a function of RNA concentration, one can detect the concentration range wherein increased fluorescence is the result of dimerization.

Base Sequence↗

PubMed1983

Magnesium ion induced proton release as a probe for the polyelectrolytic structure of ribosomal RNAs and subunits.

E coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1 mM to 20 mM. The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method. The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot. From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding. The subunits and rRNA's, however, differed in the proton release. 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit. The marked difference between the two subunits suggest that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents. The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association. E. coli tRNA and poly U also showed Mg2+-induced proton release. The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded. The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper. It also reflects the conformational structure of each RNA molecule.

Escherichia coli↗

PubMed1980

Role of interaction energy in the specificity of transcription. II-The Watson Crick A-U base pair template.

In continuation with work regarding the evaluation of the energy of association of various RNA bases with various base pairs, the results of the computations of the electrostatic interaction energy of A-U base pair with four RNA bases viz. Adenine, Uracil, Guanine and Cytosine have been reported. Non bonded induced polarization and dispersion potentials are not taken into account. Electrostatic hard sphere model of Nash and Bradley has been employed. Computations have been performed to find out the minimum energy configuration out of the various possible complex configurations. Results have been discussed with reference to similar calculations with G-C base pair performed by the authors.

Adenine↗

PubMed2003

Crystal structure of the 2'-5' RNA ligase from Thermus thermophilus HB8.

The 2'-5' RNA ligase family members are bacterial and archaeal RNA ligases that ligate 5' and 3' half-tRNA molecules with 2',3'-cyclic phosphate and 5'-hydroxyl termini, respectively, to the product containing the 2'-5' phosphodiester linkage. Here, the crystal structure of the 2'-5' RNA ligase protein from an extreme thermophile, Thermus thermophilus HB8, was solved at 2.5A resolution. The structure of the 2'-5' RNA ligase superimposes well on that of the Arabidopsis thaliana cyclic phosphodiesterase (CPDase), which hydrolyzes ADP-ribose 1",2"-cyclic phosphate (a product of the tRNA splicing reaction) to the monoester ADP-ribose 1"-phosphate. Although the sequence identity between the two proteins is remarkably low (9.3%), the 2'-5' RNA ligase and CPDase structures have two HX(T/S)X motifs in their corresponding positions. The HX(T/S)X motifs play important roles in the CPDase activity, and are conserved in both the CPDases and 2'-5' RNA ligases. Therefore, the catalytic mechanism of the 2'-5' RNA ligase may be similar to that of the CPDase. On the other hand, the electrostatic potential of the cavity of the 2'-5' RNA ligase is positive, but that of the CPDase is negative. Furthermore, in the CPDase, two loops with low B-factors cover the cavity. In contrast, in the 2'-5' RNA ligase, the corresponding loops form an open conformation and are flexible. These characteristics may be due to the differences in the substrates, tRNA and ADP-ribose 1",2"-cyclic phosphate.

Amino Acid Motifs↗

PubMedDate not supplied

Three-dimensional model of a selective theophylline-binding RNA molecule.

A three-dimensional (3D) model for an RNA molecule that selectively binds theophylline but not caffeine is proposed. This RNA, which was found using SELEX (Jenison et al., 1994), is 10,000 times more specific for theophylline (Kn = 320 nM) than for caffeine (KD = 3.5 mM), although the two ligands are identical except for a methyl group substituted at N7 (present only in caffeine). The binding affinity for ten xanthine-based ligands was used to derive a comparative molecular field analysis model (R2 = 0.93 for three components, with cross-validated R2 of 0.73), using the SYBYL and GOLPE programs. A pharmacophoric map was generated to locate steric and electrostatic interactions between theophylline and the RNA binding site. This information was used to identify putative functional groups of the binding pocket and to generate distance constraints. On the basis of a model for the secondary structure (Jenison et al., 1994), the 3D structure of this RNA was then generated using the following method: each helical region of the RNA molecule was treated as a rigid body; single-stranded loops with specific end-to-end distances were generated. The structures of RNA-xanthine complexes were studied using a modified Monte Carlo algorithm. The detailed structure of an RNA-ligand complex model, as well as possible explanations for the theophylline selectivity are discussed.

Base Sequence↗

PubMed2001

Two functionally distinct steps mediate high affinity binding of U1A protein to U1 hairpin II RNA.

Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A.U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electrostatic interactions and a subsequent locking step based on close-range interactions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for high affinity RNA/protein interactions.

Amino Acid Sequence↗

PubMed2005

The RNA-bound conformation of neamine as determined by transferred NOE experiments.

The tRNA(Phe)-bound conformation of the aminoglycoside neamine, a member of the neomycin B family, has been investigated by transferred NOE experiments in aqueous solution. This is the first time that the bioactive conformation of an RNA-bound aminoglycoside has been determined by this method. In buffers without divalent Mg(2+) ions, a high degree of electrostatically driven unspecific binding of aminoglycosides to the RNA was observed. Careful optimization of experimental conditions yielded buffer conditions optimized for cryo-probe NMR experiments. In particular, addition of Mg(2+) ions to the solutions was necessary to reduce the amount of unspecific binding as monitored by one-dimensional NMR and surface plasmon resonance experiments. CD spectroscopy was used to probe the effect of aminoglycosides and buffer conditions on the double helical content of tRNA(Phe). Finally the tRNA(Phe)-bound conformation of neamine was determined by trNOE build-up curves and compared with the previously reported crystal structure of neomycin B complexed to this RNA. Although the aminoglycoside in the crystal structure contains several configurational errors, the overall shape of the crystallographically determined RNA-bound structure is identical to the RNA-bound conformation defined by the NMR experiments. Therefore, the crystal structure has been refined by trNOE data. This is particularly important in the context of aminoglycosides being discussed as lead structures for the development of new anti-RNA drugs.

Aminoglycosides↗

PubMed2001

The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core.

Sm proteins form the core of small nuclear ribonucleoprotein particles (snRNPs), making them key components of several mRNA-processing assemblies, including the spliceosome. We report the 1.75-A crystal structure of SmAP, an Sm-like archaeal protein that forms a heptameric ring perforated by a cationic pore. In addition to providing direct evidence for such an assembly in eukaryotic snRNPs, this structure (i) shows that SmAP homodimers are structurally similar to human Sm heterodimers, (ii) supports a gene duplication model of Sm protein evolution, and (iii) offers a model of SmAP bound to single-stranded RNA (ssRNA) that explains Sm binding-site specificity. The pronounced electrostatic asymmetry of the SmAP surface imparts directionality to putative SmAP-RNA interactions.

Amino Acid Sequence↗

PubMed2003

Microenvironment analysis and identification of magnesium binding sites in RNA.

Interactions with magnesium (Mg2+) ions are essential for RNA folding and function. The locations and function of bound Mg2+ ions are difficult to characterize both experimentally and computationally. In particular, the P456 domain of the Tetrahymena thermophila group I intron, and a 58 nt 23s rRNA from Escherichia coli have been important systems for studying the role of Mg2+ binding in RNA, but characteristics of all the binding sites remain unclear. We therefore investigated the Mg2+ binding capabilities of these RNA systems using a computational approach to identify and further characterize their Mg2+ binding sites. The approach is based on the FEATURE algorithm, reported previously for microenvironment analysis of protein functional sites. We have determined novel physicochemical descriptions of site-bound and diffusely bound Mg2+ ions in RNA that are useful for prediction. Electrostatic calculations using the Non-Linear Poisson Boltzmann (NLPB) equation provided further evidence for the locations of site-bound ions. We confirmed the locations of experimentally determined sites and further differentiated between classes of ion binding. We also identified potentially important, high scoring sites in the group I intron that are not currently annotated as Mg2+ binding sites. We note their potential function and believe they deserve experimental follow-up.

Algorithms↗

PubMed2002

The GAAA tetraloop-receptor interaction contributes differentially to folding thermodynamics and kinetics for the P4-P6 RNA domain.

Tetraloops with the generic sequence GNRA are commonly found in RNA secondary structure, and interactions of such tetraloops with "receptors" elsewhere in RNA play important roles in RNA structure and folding. However, the contributions of tetraloop-receptor interactions specifically to the kinetics of RNA tertiary folding, rather than the thermodynamics of maintaining tertiary structure once folded, have not been reported. Here we investigate the role of the key GAAA tetraloop-receptor motif in folding of the P4-P6 domain of the Tetrahymena group I intron RNA. Insertions of one or more nucleotides into the tetraloop significantly disrupt the thermodynamics of tertiary folding; single-nucleotide insertions shift the folding free energy by 2-4 kcal/mol (DeltaDeltaG(o)'). The folding kinetics of several modified P4-P6 domains were determined by stopped-flow fluorescence spectroscopy, using an internally incorporated pyrene residue as the chromophore. In contrast to the thermodynamic results, the kinetics of Mg(2+)-induced folding were barely affected by the tetraloop modifications, with a DeltaDeltaG(++) of 0.2-0.4 kcal/mol and a Phi value (ratio of the kinetic and thermodynamic contributions) of <0.1. These data indicate an early transition state for folding of P4-P6 with respect to forming the tetraloop-receptor contact, consistent with previous results for modifications elsewhere in P4-P6. We conclude that the GAAA tetraloop-receptor motif contributes little to the stabilization of the transition state for Mg(2+)-induced P4-P6 folding. Rather, the tetraloop-receptor motif acts to clamp the RNA once folding has occurred. This is the first report to correlate the kinetic and thermodynamic contributions of an important RNA tertiary motif, the GNRA tetraloop-receptor. The results are related to possible models for the Mg(2+)-induced folding of the P4-P6 RNA, including a model invoking rapid nonspecific electrostatic collapse.

Adenine↗

PubMed2004

Acid-base versus structural properties of an aminoglycoside antibiotic--sisomicin: NMR and potentiometric approach.

Aminoglycoside antibiotics constitute a class of the drugs of high interest, whose therapeutic action is based upon the electrostatic interaction with the variety of RNA molecules. The positive charge of these drugs molecules, located at their amino functions, has a prevailing influence on this process. The potentiometry and (1)H NMR spectroscopy are applied hereby to achieve the characteristics of the acid-base properties of particular protonating groups. We found that the pK values of deprotonation processes cover a wide values range 6-9.8. The correlation spectra of sisomicin, both COSY and TOCSY, allowed attributing unambiguously individual signals to the corresponding protons. These spectra involve a lot of the cross-peaks originating from the B and C rings protons, while the analogous signals originating from A rings protons are less numerous. Molecular modeling provided that the methylated amino group of A ring is located too far from the protonated functions of the remaining rings to affect their pK values. The phenomena observed herein are discussed in line of strength of the analogous processes observed for other aminoglycosides. As the result, four types of amino groups consisted within these antibiotics are distinguished.

Acid-Base Equilibrium↗