Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion

Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop–loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug–RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K_d ~ 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K_d ~ 1.6 µM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop–loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.     

Determination of thermodynamic parameters for HIV DIS type loop-loop kissing complexes

The HIV-1 type dimerization initiation signal (DIS) loop was used as a starting point for the analysis of the stability of Watson–Crick (WC) base pairs in a tertiary structure context. We used ultraviolet melting to determine thermodynamic parameters for loop–loop tertiary interactions and compared them with regular secondary structure RNA helices of the same sequences. In 1 M Na⁺ the loop–loop interaction of a HIV-1 DIS type pairing is 4 kcal/mol more stable than its sequence in an equivalent regular and isolated RNA helix. This difference is constant and sequence independent, suggesting that the rules governing the stability of WC base pairs in the secondary structure context are also valid for WC base pairs in the tertiary structure context. Moreover, the effect of ion concentration on the stability of loop–loop tertiary interactions differs considerably from that of regular RNA helices. The stabilization by Na⁺ and Mg²⁺ is significantly greater if the base pairing occurs within the context of a loop–loop interaction. The dependence of the structural stability on salt concentration was defined via the slope of a T_m/log [ion] plot. The short base-paired helices are stabilized by 8°C/log [Mg²⁺] or 11°C/log [Na⁺], whereas base-paired helices forming tertiary loop–loop interactions are stabilized by 16°C/log [Mg²⁺] and 26°C/log [Na⁺]. The different dependence on ionic strength that is observed might reflect the contribution of specific divalent ion binding to the preformation of the hairpin loops poised for the tertiary kissing loop–loop contacts.     

Identification of antisense RNA stem–loops that inhibit RNA–protein interactions using a bacterial reporter system

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem–loop RNAs, presumably due to the high thermodynamic stability of the resulting loop–loop and loop–linear interactions. In this study, the identification of RNA stem–loops that inhibit U1A protein binding to the hpII RNA through RNA–RNA interactions was attempted using a bacterial reporter system based on phage λ N-mediated antitermination. As a result, loop sequences possessing 7–8 base complementarity to the 5′ region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem–loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem–loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem–loops that disrupt various types of RNA–protein interactions.     

Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes.     

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.     

A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme

Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem–loop I (SLI) substrate and stem–loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem–loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8–3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7–8 kcal/mol than predicted for a comparable duplex containing three Watson–Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6–7 Watson–Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2–3 Watson–Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.     

Structure–function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop–loop interaction motif conserved in most natural hammerheads

Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5′ side and an extrahelical U at its 3′-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5′ U of loop 1 and the 3′ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.     

Stabilities of HIV-1 DIS type RNA loop-loop interactions in vitro and in vivo

RNA loop–loop interactions are a prevalent motif in the formation of tertiary structure and are well suited to trigger molecular recognition between RNA molecules. We determined the stabilities of several loop–loop interactions with a constant 6 bp core sequence and varying unpaired flanking nucleotides and found that the flanking bases have a strong influence on the stability and ion dependence of the kissing complex. In general, the stabilities determined in 1 M Na⁺ are equivalent to those in the presence of near physiological Mg²⁺ concentrations. Therefore we further tested whether the stabilities determined in vitro and within yeast cells correlate, using a recently developed yeast RNA-hybrid system. For the majority of the loop types analyzed here, the melting temperatures determined in vitro are in good agreement with the relative β-galactosidase activity in yeast cells, showing that data derived from in vitro measurements reflect in vivo properties. The most stable interactions are the naturally occurring HIV-1 DIS MAL and LAI derived loops with the motif (5′ A^A/_GN₆A 3′), emphasizing the crucial role of stable kissing complexes in HIV genome dimerization.     

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.     

RNA aptamers that specifically bind to a 16S ribosomal RNA decoding region construct

RNA–RNA recognition is a critical process in controlling many key biological events, such as translation and ribozyme functions. The recognition process governing RNA–RNA interactions can involve complementary Watson–Crick (WC) base pair binding, or can involve binding through tertiary structural interaction. Hence, it is of interest to determine which of the RNA–RNA binding events might emerge through an in vitro selection process. The A-site of the 16S rRNA decoding region was chosen as the target, both because it possesses several different RNA structural motifs, and because it is the rRNA site where codon/anticodon recognition occurs requiring recognition of both mRNA and tRNA. It is shown here that a single family of RNA molecules can be readily selected from two different sizes of RNA library. The tightest binding aptamer to the A-site 16S rRNA construct, 109.2-3, has its consensus sequences confined to a stem–loop region, which contains three nucleotides complementary to three of the four nucleotides in the stem–loop region of the A-site 16S rRNA. Point mutations on each of the three nucleotides on the stem–loop of the aptamer abolish its binding capacity. These studies suggest that the RNA aptamer 109.2-3 interacts with the simple 27 nt A-site decoding region of 16S rRNA through their respective stem–loops. The most probable mode of interaction is through complementary WC base pairing, commonly referred to as a loop–loop ‘kissing’ motif. High affinity binding to the other structural motifs in the decoding region were not observed.     

HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway

The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5′-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a ‘kissing complex’ between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg²⁺, but not by K⁺ cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg²⁺ ions.     

Putative intermediary stages for the molecular evolution from a ribozyme to a catalytic RNP

A hypothetical evolutionary pathway from a ribozyme to a catalytic RNA–protein complex (RNP) is proposed and examined. In this hypothesis for an early phase of molecular evolution, one RNA–RNA interaction in the starting ribozyme is replaced with an RNA–protein interaction via two intermediary stages. At each stage, the original RNA–RNA interaction and a newly introduced RNA–protein interaction are designed to coexist. The catalytic RNPs corresponding to the intermediary stages were constructed by employing the Tetrahymena ribozyme together with molecular modeling. Analyses of the RNPs indicate that the protein can fully replace the original role of the RNA–RNA interaction in the starting ribozyme and that the association of a protein with a ribozyme might be beneficial for improving the ribozymatic activity.     

Conserved tertiary base pairing ensures proper RNA folding and efficient assembly of the signal recognition particle Alu domain

Proper folding of the RNA is an essential step in the assembly of functional ribonucleoprotein complexes. We examined the role of conserved base pairs formed between two distant loops in the Alu portion of the mammalian signal recognition particle RNA (SRP RNA) in SRP assembly and functions. Mutations disrupting base pairing interfere with folding of the Alu portion of the SRP RNA as monitored by probing the RNA structure and the binding of the protein SRP9/14. Complementary mutations rescue the defect establishing a role of the tertiary loop–loop interaction in RNA folding. The same mutations in the Alu domain have no major effect on binding of proteins to the S domain suggesting that the S domain can fold independently. Once assembled into a complete SRP, even particles that contain mutant RNA are active in arresting nascent chain elongation and translocation into microsomes, and, therefore, tertiary base pairing does not appear to be essential for these activities. Our results suggest a model in which the loop–loop interaction and binding of the protein SRP9/14 play an important role in the early steps of SRP RNA folding and assembly.     

Molecular dynamics reveals the stabilizing role of loop closing residues in kissing interactions: comparison between TAR-TAR* and TAR-aptamer

A RNA aptamer (R06) raised against the trans- activation responsive (TAR) element of HIV-1 was previously shown to generate a loop–loop complex whose stability is strongly dependent on the selected G and A residues closing the aptamer loop. The rationally designed TAR* RNA hairpin with a loop sequence fully complementary to the TAR element, closed by U,A residues, also engages in a loop–loop association with TAR, but with a lower stability compared with the TAR–R06 complex. UV absorption monitored thermal denaturation showed that TAR–TAR*(GA), in which the U,A kissing residues were exchanged for G,A, is as stable as the selected TAR–R06 complex. Consequently, we used the TAR–TAR* structure deduced from NMR studies to model the TAR–R06 complex with either GA, CA or UA loop closing residues. The results of the molecular dynamics trajectories correlate well with the thermal denaturation experiments and show that the increased stability of the GA variant results from an optimized stacking of the bases at the stem–loop junction and from stable interbackbone hydrogen bonds.     

Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson–Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson–Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson–Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3′ or 5′ of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.     

Predicting structure and stability for RNA complexes with intermolecular loop–loop base-pairing

RNA loop–loop interactions are essential for genomic RNA dimerization and regulation of gene expression. In this article, a statistical mechanics-based computational method that predicts the structures and thermodynamic stabilities of RNA complexes with loop–loop kissing interactions is described. The method accounts for the entropy changes for the formation of loop–loop interactions, which is a notable advancement that other computational models have neglected. Benchmark tests with several experimentally validated systems show that the inclusion of the entropy parameters can indeed improve predictions for RNA complexes. Furthermore, the method can predict not only the native structures of RNA/RNA complexes but also alternative metastable structures. For instance, the model predicts that the SL1 domain of HIV-1 RNA can form two different dimer structures with similar stabilities. The prediction is consistent with experimental observation. In addition, the model predicts two different binding sites for hTR dimerization: One binding site has been experimentally proposed, and the other structure, which has a higher stability, is structurally feasible and needs further experimental validation.     

Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B. subtilis RNase P protein

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5′-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain–P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of ~0.1 µM. The C-domain–P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (~0.9 min^–1 at pH 7.8) and substrates with a hairpin–loop 3′ to the cleavage site (~40 min^–1). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10–500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem–loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.     

Identification of a new RNA.RNA interaction site for human telomerase RNA (hTR): structural implications for hTR accumulation and a dyskeratosis congenita point mutation

The enzyme telomerase is a ribonucleoprotein that has a critical role in the maintenance of stable telomeres in organisms that possess linear chromosomes. Using a recently developed single molecule fluorescence coincidence method, we have studied the RNA component of telomerase (hTR) and directly observed multimerisation of hTR in solution. RNA mutagenesis and blocking oligonucleotides were employed to identify the single-stranded internal loop J7b/8a as an important and specific hTR·hTR interaction site. This observation was confirmed by studies on a model RNA fragment (hTR_380–444), comprising part of the H/ACA domain, the internal loop J7b/8a and the CR7 domain, that was found to dimerise. Substitution mutagenesis within the proposed RNA·RNA interaction site of hTR_380–444 resulted in a loss of dimerisation potential and insertion of the dyskeratosis congenita mutation C408G led to a significant reduction in dimer formation. Together, these results suggest that this RNA·RNA interaction site may be functionally relevant.     

Discriminating duplex and hairpin oligonucleotides using chemical shifts: application to the anticodon stem–loop of Escherichia coli tRNAPhe

A sensitive NMR spectroscopic method for detection of duplex forms of self-complementary nucleic acid sequences has been implemented. The G·U wobble base pair formed between a ¹⁵N-labeled strand and an unlabeled probe strand is used to identify the duplex. The guanine imino resonance, with its characteristic chemical shift, is detected using a 2D ¹⁵N–¹H heteronuclear multiple quantum coherence (HMQC) spectrum and provides a sensitive and unambiguous route to hairpin–duplex discrimination. The method has been used to identify the duplex and hairpin forms of an RNA oligonucleotide at concentrations of ~20 µM. This method has also been used to rule out possible duplex formation of an RNA oligonucleotide corresponding to the unmodified anticodon stem–loop of Escherichia coli tRNA^Phe and suggests that this hairpin has a 3 nt loop.