Structural basis of ligand discrimination by two related RNA aptamers resolved by NMR spectroscopy.

In a previous study, an RNA aptamer for the specific recognition of arginine was evolved from a parent sequence that bound citrulline specifically. The two RNAs differ at only 3 positions out of 44. The solution structures of the two aptamers complexed to their cognate amino acids have now been determined by two-dimensional nuclear magnetic resonance spectroscopy. Both aptamers contain two asymmetrical internal loops that are not well ordered in the free RNA but that fold into a compact structure upon ligand binding. Those nucleotides common to both RNAs include a conserved cluster of purine residues, three of which form an uneven plane containing a G:G pair, and two other residues nearly perpendicular to that surface. Two of the three variant nucleotides are stacked on the cluster of purines and form a triple contact to the amino acid side chain, whereas the edge of the third variant nucleotide is capping the binding pocket.

A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements.

For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative spatial orientation of the three constituent Watson-Crick base-paired helical segments. Synthetic constructs were labeled with the fluorescence donor (5-carboxyfluorescein) and acceptor (5-carboxytetramethylrhodamine) located at the ends of the strands constituting the ribozyme molecule. The acceptor helix in helix pairs I and III and in II and III was varied in length from 5 to 11 and 5 to 9 base pairs, respectively, and the FRET efficiencies were determined and correlated with a reference set of labeled RNA duplexes. The FRET efficiencies were predicted on the basis of vector algebra analysis, as a function of the relative helical orientations in the ribozyme constructs, and compared with experimental values. The data were consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. These orientational constraints were used for molecular modeling of a three-dimensional structure of the complete ribozyme.

The interaction networks of structured RNAs.

All pairwise interactions occurring between bases which could be detected in three-dimensional structures of crystallized RNA molecules are annotated on new planar diagrams. The diagrams attempt to map the underlying complex networks of base-base interactions and, especially, they aim at conveying key relationships between helical domains: co-axial stacking, bending and all Watson-Crick as well as non-Watson-Crick base pairs. Although such wiring diagrams cannot replace full stereographic images for correct spatial understanding and representation, they reveal structural similarities as well as the conserved patterns and distances between motifs which are present within the interaction networks of folded RNAs of similar or unrelated functions. Finally, the diagrams could help devising methods for meaningfully transforming RNA structures into graphs amenable to network analysis.

A three-dimensional model of hepatitis delta virus ribozyme based on biochemical and mutational analyses.

BACKGROUND: Hepatitis delta virus (HDV), which has a single-stranded RNA genome about 1700 nucleotides long, is a satellite virus of hepatitis B, and is associated with a high incidence of fulminant hepatitis and death in infected humans. Like certain pathogenic subviral RNAs that infect plants, HDV RNA features a closed-circular conformation, a rolling-circle mechanism of replication and RNA-catalyzed self-cleaving reactions of both genomic and anti-genomic strands in vitro. The catalytic domains cannot be folded into either the hammerhead or hairpin secondary-structure motifs that have been found in other self-cleaving RNAs. RESULTS: A pseudoknot secondary-structure model has been suggested for the catalytic domain (ribozyme) of HDV RNA. We conducted extensive mutational analyses of regions of the HDV ribozyme predicted in this model to be single stranded, and found that several of them are important for catalytic activity. We used these data, sequence comparisons between different isolates and previously published structural analyses to produce a computer graphic model of the three-dimensional architecture of the HDV ribozyme. CONCLUSIONS: Our model supports the pseudoknotted structure and rationalizes several observations relating to the lengths of the various stems and the sequence requirements of the single-stranded regions. It also provides insight into the catalytic mechanism of the HDV ribozyme. We specifically propose that residues C75, U20 and C21 form the basis of the catalytic region and are close to the cleavable phosphate.

Simulations of the molecular dynamics of nucleic acids.

The growing amount of high quality molecular dynamics simulations generated using the latest methodological developments and force fields has led to a sharper understanding of the forces underlying the dynamics of biomolecular systems, as well as to stimulating insights into the structure and catalysis of nucleic acids. It is now clear that inclusion of long-range electrostatic interactions and of the aqueous and ionic environment is necessary for producing realistic and accurate simulations. Yet, many papers hint at a force field and protocol dependence of the results and thus contain the seeds for the future improvements that will be necessary for deepening our understanding of recognition phenomena and folding of nucleic acids.

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.

The A-minor motifs in the decoding recognition process.

The formation of A-minor motifs, mediated by adenines binding into the shallow/minor groove of stacked and helical Watson-Crick base pairs, is described. The conformations of the bacterial ribosomal decoding A site in various crystal structures are reviewed. The adenines A1492 and A1493 of the A site are seen either tucked in within the internal loop or bulging out and poised for interaction. This dynamic equilibrium contributes to the decoding process of the codon:anticodon base pairings. Aminoglycoside antibiotics lock the conformation of the A site in a single state with bulged-out adenines and thereby disrupt regulation of the decoding process.

A molecular dynamics simulation study of an aminoglycoside/A-site RNA complex: conformational and hydration patterns.

Aminoglycoside antibiotics interfere with the translation mechanism by binding to the tRNA decoding site of the 16S ribosomal RNA. Crystallographic structures of aminoglycosides bound to A-site systems clarified many static aspects of RNA-ligand interactions. To gain some insight on the dynamic aspects of recognition phenomena, we conducted molecular dynamics simulations of the aminoglycoside paromomycin bound to a eubacterial ribosomal decoding A-site oligonucleotide. Results from 25 ns of simulation time revealed that: (i) the neamine part of the antibiotic represents the main anchor for binding, (ii) additional sugar rings provide limited and fragile contacts, (iii) long-resident water molecules present at the drug/RNA interface are involved in the recognition phenomena. The combination of MD simulations together with systematic structural information offers striking insights into the molecular recognition processes underlying RNA/aminoglycoside binding. Important methodological considerations related to the use of medium resolution starting structures and associated sampling problems are thoroughly discussed.

Mutagenesis of 16S rRNA C1409-G1491 base-pair differentiates between 6'OH and 6'NH3+ aminoglycosides.

Using a single rRNA allelic Gram-positive model system, we systematically mutagenized 16S rRNA positions 1409 and 1491 to probe the functional relevance of structural interactions between aminoglycoside antibiotics and the A-site rRNA that were suggested by X-ray crystallography. At the structural level, the interaction of the 2-deoxystreptamine aminoglycosides with the rRNA base-pair C1409-G1491 has been suggested to involve the following features: (i) ring I of the disubstituted 2-deoxystreptamines stacks upon G1491 and H-bonds to the Watson-Crick edge of A1408; (ii) ring III of the 4,5-disubstituted aminoglycosides shows hydrogen bonding to G1491. However, we found that mutants with altered 16S rRNA bases 1409 and 1491 discriminated poorly between 4,5-disubstituted and 4,6-disubstituted 2-deoxystreptamines, but differentially affected aminoglycosides with a hydroxyl group versus an ammonium group at position 6' of ring I, e.g. G1491U conferred high-level drug resistance to paromomycin and geneticin, but not to neomycin, tobramycin or gentamicin.

TectoRNA: modular assembly units for the construction of RNA nano-objects.

Structural information on complex biological RNA molecules can be exploited to design tectoRNAs or artificial modular RNA units that can self-assemble through tertiary interactions thereby forming nanoscale RNA objects. The selective interactions of hairpin tetraloops with their receptors can be used to mediate tectoRNA assembly. Here we report on the modulation of the specificity and the strength of tectoRNA assembly (in the nanomolar to micromolar range) by variation of the length of the RNA subunits, the nature of their interacting motifs and the degree of flexibility of linker regions incorporated into the molecules. The association is also dependent on the concentration of magnesium. Monitoring of tectoRNA assembly by lead(II) cleavage protection indicates that some degree of structural flexibility is required for optimal binding. With tectoRNAs one can compare the binding affinities of different tertiary motifs and quantify the strength of individual interactions. Furthermore, in analogy to the synthons used in organic chemistry to synthesize more complex organic compounds, tectoRNAs form the basic assembly units for constructing complex RNA structures on the nanometer scale. Thus, tectoRNA provides a means for constructing molecular scaffoldings that organize functional modules in three-dimensional space for a wide range of applications.

Phylogenetic analysis of tmRNA genes within a bacterial subgroup reveals a specific structural signature.

Bacterial tmRNA mediates a trans-translation reaction, which permits the recycling of stalled ribosomes and probably also contributes to the regulated expression of a subset of genes. Its action results in the addition of a small number of C-terminal amino acids to protein whose synthesis had stalled and these constitute a proteolytic recognition tag for the degradation of these incompletely synthesized proteins. Previous work has identified pseudoknots and stem-loops that are widely conserved in divergent bacteria. In the present work an alignment of tmRNA gene sequences within 13 beta-proteobacteria reveals an additional sub-structure specific for this bacterial group. This sub-structure is in pseudoknot Pk2, and consists of one to two additional stem-loop(s) capped by stable GNRA tetraloop(s). Three-dimensional models of tmRNA pseudoknot 2 (Pk2) containing various topological versions of the additional sub-structure suggest that the sub-structures likely point away from the core of the RNA, containing both the tRNA and the mRNA domains. A putative tertiary interaction has also been identified.

Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex.

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA-CopT complexes suggests that, subsequent to loop-loop contact, helix propagation is prevented. Instead, a fully base paired loop-loop interaction is formed, inducing a continuous stacking of three helices. Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the four-way junction topology, the loop-loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.

RNA folding: beyond Watson-Crick pairs.

Several crystal structures of RNA fragments, alone or in complex with a specific protein, have been recently solved. In addition, the structures of an artificial ribozyme, the leadzyme, and the cleavage product of a human pathogen ribozyme, have extended the structural diversity of ribozyme architectures. The attained set of folding rules and motifs expand the repertoire seen previously in tRNA structures.

Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site.

BACKGROUND: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA. RESULTS: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 A resolution. Each RNA fragment contains two A sites inserted between Watson-Crick pairs. The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines. In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site. In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics. Ring I of paromomycin stacks over base G1491 and forms pseudo-Watson-Crick contacts with A1408. Both the hydroxyl group and one ammonium group of ring II form direct and water-mediated hydrogen bonds to the U1495oU1406 pair. The bulging conformation of the two adenines A1492 and A1493 is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II. The hydrophilic sites of the bulging A1492 and A1493 contact the shallow groove of G=C pairs in a symmetrical complex. CONCLUSIONS: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.

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

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

Somatic mutation in human T-cell leukemia virus type 1 provirus and flanking cellular sequences during clonal expansion in vivo.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma, shows intrapatient genetic variability. Although HTLV-1 can replicate via the reverse transcription of virion RNA to a double-stranded DNA provirus (the conventional manner for retroviruses), its predominant mode of replication is via the clonal expansion (mitosis) of the infected cell. This expansion is achieved by the viral oncoprotein Tax, which keeps the infected CD4 T lymphocyte cycling. Because Tax also interferes with cellular DNA repair pathways, we investigated whether somatic mutations of the provirus that occur during the division of infected cells could account for HTLV-1 genetic variability. METHODS: An inverse polymerase chain reaction strategy was designed to distinguish somatic mutations from reverse transcription-associated substitutions. This strategy allows the proviral sequences to be isolated together with flanking cellular sequences. Using this method, we sequenced 208 HTLV-1 provirus 3' segments, together with their integration sites, belonging to 29 distinct circulating cellular clones from infected individuals. RESULTS: For 60% of the clones, 8%-80% of infected cells harbored a mutated HTLV-1 provirus, without evidence of reverse transcription-associated mutations. Mutations within flanking cellular sequences were also identified at a frequency of 2.8 x 10(-4) substitution per base pair. Some of these clones carried multiple discrete substitutions or deletions, indicating progressive accumulation of mutations during clonal expansion. The overall frequency of somatic mutations increased with the degree of proliferation of infected T cells. CONCLUSIONS: These data indicate that, in vivo, HTLV-1 variation results mainly from postintegration events that consist of somatic mutations of the proviral sequence occurring during clonal expansion. The finding of substitutions in flanking sequences suggests that somatic mutations occurring after integration, presumably coupled with selection, help move the cellular clones toward a transformed phenotype, of which adult T-cell leukemia/lymphoma is the end point.

Search of essential parameters for the aminoacylation of viral tRNA-like molecules. Comparison with canonical transfer RNAs.

Comparative structural and functional results on the valine and tyrosine accepting tRNA-like molecules from turnip yellow mosaic virus (TYMV) and brome mosaic virus (BMV), and the corresponding cognate yeast tRNAs are presented. Novel experiments on TYMV RNA include design of variant genes of the tRNA-like domain and their transcription in vitro by T7 RNA polymerase, analysis of their valylation catalyzed by yeast valyl-tRNA synthetase, and structural mapping with dimethyl sulfate and carbodiimide combined with graphical modelling. Particular emphasis is given to conformational effects affecting the valylation capacity of the TYMV tRNA-like molecule (e.g., the effect of the U43----C43 mutation). The contacts of the TYMV and BMV RNAs with valyl- and tyrosyl-tRNA synthetases are compared with the positions in the molecules affecting their aminoacylation capacities. Finally, the involvement of the putative valine and tyrosine anticodons in the tRNA-like valylation and tyrosylation reactions is discussed. While an anticodon-like sequence participates in the valine identity of TYMV RNA, this seems not to be the case for the tyrosine identity of BMV RNA despite the fact that the tyrosine anticodon has been shown to be involved in the tyrosylation of canonical tRNA.

The conformation of the initiator tRNA and of the 16S rRNA from Escherichia coli during the formation of the 30S initiation complex.

The conformation of the E. coli initiator tRNA and of the 16S rRNA at different steps leading to the 30S.IF2.fMet-ARN(fMet).AUG.GTP complex has been investigated using several structure-specific probes. As compared to elongator tRNA, the initiator tRNA exhibits specific structural features in the anticodon arm, the T and D loops and the acceptor arm. Initiation factor 2 (IF2) interacts with the T-loop and the minor groove of the T stem of the RNA, and induces an increased flexibility in the anticodon arm. In the 30S initiation complex, additional protection is observed in the acceptor stem and in the anticodon arm of the tRNA. Within the 30S subunit, IF2 does not significantly shield defined portions of 16S rRNA, but induces both reduction and enhancement of reactivity scattered in the entire molecule. Most are constrained in a region corresponding to the cleft, the lateral protrusion and the part of the head facing the protrusion. All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG message. The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding.

Aminoglycoside-RNA interactions.

The structural and physico-chemical parameters promoting the binding of aminoglycosides to RNAs are becoming clear. The strength of the interaction is dominated by electrostatics, with the positively charged aminoglycosides displacing metal ions. Although aminoglycosides inhibit most known ribozymes, aminoglycosides or polyamines are able to catalyze specific RNA cleavage in the absence of metal ions.

Rules governing the orientation of the 2'-hydroxyl group in RNA.

Molecular dynamics simulations reveal that, in C3'-endo sugar puckers, only three orientations are accessible to the 2'-hydroxyl groups distinctive of RNA molecules: towards (i) the O3', (ii) the O4' of the same sugar, and (iii) the shallow groove base atoms. In the rarer C2'-endo sugar puckers, orientations towards the O3' atom of the same sugar are strongly favoured. Surprisingly, in helical regions, the frequently suggested intra-strand O2'-H(n)...O4'(n+1) interaction is not found. This observation led to the detection of an axial C-H...O interaction between the C2'-H2'(n) group and the O4'(n+1) atom contributing to the stabilization of RNA helical regions. Subsequent analysis of crystallographic structures of both RNA and A-DNA helices fully supports this finding. Specific hydration patterns are also thought to play a significant role in the stabilization of RNA structures. In the shallow groove of RNA, known as a favourable RNA or protein-binding region, three well-defined hydration sites are located around the O2' atom. These hydration sites, occupied by water molecules exchanging with the bulk, constitute, after dehydration, anchor points for specific interactions between RNA and nucleic acids, proteins or drugs. Therefore, the fact that the 2'-hydroxyl group is not monopolised by axial stabilization, together with its water-like behaviour, facilitates complex formation involving RNA helical regions.