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N6-methyladenosine (m6A) is the most abundant inner RNA modification in eukaryotes. Due to the development of RNA sequencing technology, the distribution pattern of m6A in the transcriptome has been uncovered. Dynamically, the reversible N6-methylation is mediated by two types of proteins, which are classified as “writers” and “erasers”. Under the association of specific co-factors, writers show spatiotemporal N6-methyltransferase activity. Mechanically, m6A can be recognized by “reader” proteins or can directly modify RNA conformation, and it widely affects gene expression by mediating RNA stability, translation, splicing and export. m6A is involved in a series of physiology processes. Dysregulation of m6A is gradually defined as the pathogenesis of some diseases, e.g., cancer and cardiovascular disease. Therefore, a good understanding of m6A is essential for molecular biology and pathology research. In this article we systemically present an overview of the functions and mechanisms of identified m6A regulators. The discovered biological and pathological processes affected by m6A are also summarized. We hope that readers with related research interests benefit from our review.

RNA recognition frequently results in conformational changes that optimize intermolecular binding. As a consequence, the overall binding affinity of RNA to its binding partners depends not only on the intermolecular interactions formed in the bound state but also on the energy cost associated with changing the RNA conformational distribution. Measuring these “conformational penalties” is, however, challenging because bound RNA conformations tend to have equilibrium populations in the absence of the binding partner that fall outside detection by conventional biophysical methods. In this study we employ as a model system HIV-1 TAR RNA and its interaction with the ligand argininamide (ARG), a mimic of TAR’s cognate protein binding partner, the transactivator Tat. We use NMR chemical shift perturbations and relaxation dispersion in combination with Bayesian inference to develop a detailed thermodynamic model of coupled conformational change and ligand binding. Starting from a comprehensive 12-state model of the equilibrium, we estimate the energies of six distinct detectable thermodynamic states that are not accessible by currently available methods. Our approach identifies a minimum of four RNA intermediates that differ in terms of the TAR conformation and ARG occupancy. The dominant bound TAR conformation features two bound ARG ligands and has an equilibrium population in the absence of ARG that is below detection limit. Consequently, even though ARG binds to TAR with an apparent overall weak affinity [Formula: see text] , it binds the prefolded conformation with a K(d) in the nM range. Our results show that conformational penalties can be major determinants of RNA-ligand binding affinity as well as a source of binding cooperativity, with important implications for a predictive understanding of how RNA is recognized and for RNA-targeted drug discovery.

U2 snRNP is an essential component of the spliceosome. It is responsible for branch point recognition in the spliceosome A-complex via base-pairing of U2 snRNA with an intron to form the branch helix. Small molecule inhibitors target the SF3B component of the U2 snRNP and interfere with A-complex formation during spliceosome assembly. We previously found that the first SF3B inhibited-complex is less stable than A-complex and hypothesized that SF3B inhibitors interfere with U2 snRNA secondary structure changes required to form the branch helix. Using RNA chemical modifiers, we probed U2 snRNA structure in A-complex and SF3B inhibited splicing complexes. The reactivity pattern for U2 snRNA in the SF3B inhibited-complex is indistinguishable from that of A-complex, suggesting that they have the same secondary structure conformation, including the branch helix. This observation suggests SF3B inhibited-complex instability does not stem from an alternate RNA conformation and instead points to the inhibitors interfering with protein component interactions that normally stabilize U2 snRNP’s association with an intron. In addition, we probed U2 snRNA in the free U2 snRNP in the presence of SF3B inhibitor and again saw no differences. However, increased protection of nucleotides upstream of Stem I in the absence of SF3A and SF3B proteins suggests a change of secondary structure at the very 5′ end of U2 snRNA. Chemical probing of synthetic U2 snRNA in the absence of proteins results in similar protections and predicts a previously uncharacterized extension of Stem I. Because this stem must be disrupted for SF3A and SF3B proteins to stably join the snRNP, the structure has the potential to influence snRNP assembly and recycling after spliceosome disassembly.

RNA structure heterogeneity is a major challenge when querying RNA structures with chemical probing. We introduce DRACO, an algorithm for the deconvolution of coexisting RNA conformations from mutational profiling experiments. Analysis of the SARS-CoV-2 genome using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and DRACO, identifies multiple regions that fold into two mutually exclusive conformations, including a conserved structural switch in the 3' untranslated region. This work may open the way to dissecting the heterogeneity of the RNA structurome.

Pentatricopeptide repeat (PPR) proteins, a helical repeat family of organellar RNA binding proteins, play essential roles in post-transcriptional RNA processing. In Trypanosoma brucei, an expanded family of PPR proteins localize to the parasite’s single mitochondrion, where they are believed to perform important roles in both RNA processing and translation. We studied the RNA binding specificity of the simplest T. brucei PPR protein (KRIPP11) using electrophoretic mobility shift assays, fluorescence anisotropy, circular dichroism spectroscopy, and in vitro selection. We found KRIPP11 to be an RNA binding protein with specificity for sequences of four or more consecutive guanosine residues (G-tracts). Such G-tracts are dramatically enriched in T. brucei mitochondrial transcripts that are destined for extensive uridine insertion/deletion editing but are not present in mRNAs following editing. We further found that the quadruplex oligoguanosine RNA conformation is preferentially recognized by KRIPP11 over other conformational forms, and is bound without disruption of the quadruplex structure. In combination with prior data demonstrating association of KRIPP11 with the small ribosomal subunit, these results suggest possible roles for KRIPP11 in bridging mRNA maturation and translation or in facilitating translation of unusual dual-coded open reading frames.

Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given the variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely nonoverlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.

Motivation Kinetics is key to understand many phenomena involving RNAs, such as co-transcriptional folding and riboswitches. Exact out-of-equilibrium studies induce extreme computational demands, leading state-of-the-art methods to rely on approximated kinetics landscapes, obtained using sampling strategies that strive to generate the key landmarks of the landscape topology. However, such methods are impeded by a large level of redundancy within sampled sets. Such a redundancy is uninformative, and obfuscates important intermediate states, leading to an incomplete vision of RNA dynamics. Results We introduce RNANR, a new set of algorithms for the exploration of RNA kinetics landscapes at the secondary structure level. RNANR considers locally optimal structures, a reduced set of RNA conformations, in order to focus its sampling on basins in the kinetic landscape. Along with an exhaustive enumeration, RNANR implements a novel non-redundant stochastic sampling, and offers a rich array of structural parameters. Our tests on both real and random RNAs reveal that RNANR allows to generate more unique structures in a given time than its competitors, and allows a deeper exploration of kinetics landscapes. Availability and implementation RNANR is freely available at https://project.inria.fr/rnalands/rnanr.

Structured RNA elements within the internal ribosome entry site (IRES) of hepatitis C virus (HCV) genome hijack host cell machinery for translation initiation through a cap-independent mechanism. Here, using a phage display selection, we obtained two antibody fragments (Fabs), HCV2 and HCV3, against HCV IRES that bind the RNA with dissociation constants of 32 ± 7 nM and 37 ± 8 nM respectively, specifically recognizing the so-called junction IIIabc (JIIIabc). We used these Fabs as crystallization chaperones and determined the high-resolution crystal structures of JIIIabc – HCV2 and – HCV3 complexes at 1.81-Å and 2.75-Å resolution respectively, revealing an anti-parallel four-way junction with the so-called IIIa and IIIc sub-domains brought together through tertiary interactions. The RNA conformation observed in the structures supports the structural model for this region derived from cryo-EM data for the HCV IRES – 40S ribosome complex, suggesting that the tertiary fold of the RNA pre-organizes the domain for interactions with the 40S ribosome. Strikingly, both Fabs and the ribosomal protein eS27 not only interact with a common subset of nucleotides within the JIIIabc but also use physio-chemically similar sets of protein residues to do so, suggesting that the RNA surface is well-suited for interactions with proteins, perhaps analogous to the ‘hot spot’ concept elaborated for protein-protein interactions. Using a rabbit reticulocyte lysate-based translation assay with a bicistronic reporter construct, we further demonstrated that Fabs HCV2 and HCV3 specifically inhibit the HCV IRES-directed translation, implicating disruption of the JIIIabc – ribosome interaction as a potential therapeutic strategy against HCV.

Background Predicted RNA secondary structures are typically visualized using dot-plots for base pair binding probabilities and planar graphs for unique structures, such as the minimum free energy structure. These are however difficult to analyze simultaneously. Results This work introduces a compact unified view of the most stable conformation of an RNA secondary structure and its base pair probabilities, which is called the Circular Secondary Structure Base Pairs Probabilities Plot (CS 2 BP 2 -Plot). Along with our design we provide access to a web server implementation of our solution that facilitates pairwise comparison of short RNA (and DNA) sequences up to 200 base pairs. The web server first calculates the minimum free energy secondary structure and the base pair probabilities for up to 10 RNA or DNA sequences using RNAfold and then provides a two panel comparative view that includes CS2BP2-Plots along with the traditional graph, planar and circular diagrams obtained with VARNA. The CS2BP2-Plots include highlighting of the nucleotide differences between two selected sequences using ClustalW local alignments. We also provide descriptive statistics, dot-bracket secondary structure representations and ClustalW local alignments for compared sequences. Conclusions Using circular diagrams and colour and weight-coded arcs, we demonstrate how a single image can replace the state-of-the-art dual representations (dot-plots and minimum free energy structures) for base-pair probabilities of RNA secondary structures while allowing efficient exploration and comparison of different RNA conformations via a web server front end. With that, we provide the community, especially the biologically oriented, with an intuitive tool for ncRNA visualization. Web-server: https://nrcmonsrv01.nrc.ca/cs2bp2plot

Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.

Comprehensive understanding of the structure–function relationship of RNA both in real time and at atomic level will have a profound impact in advancing our understanding of RNA functions in biology. Here, we describe the first example of a multifunctional nucleoside probe, containing a conformation‐sensitive fluorophore and an anomalous X‐ray diffraction label (5‐selenophene uracil), which enables the correlation of RNA conformation and recognition under equilibrium and in 3D. The probe incorporated into the bacterial ribosomal RNA decoding site, fluorescently reports antibiotic binding and provides diffraction information in determining the structure without distorting native RNA fold. Further, by comparing solution binding data and crystal structure, we gained insight on how the probe senses ligand‐induced conformational change in RNA. Taken together, our nucleoside probe represents a new class of biophysical tool that would complement available tools for functional RNA investigations.

Three-dimensional RNA structures are notoriously difficult to determine, and the link between secondary structure and RNA conformation is only beginning to be understood. These challenges have hindered the identification of guiding principles for small molecule:RNA recognition. We herein demonstrate that the strong and differential binding ability of aminoglycosides to RNA structures can be used to classify five canonical RNA secondary structure motifs through principal component analysis (PCA). In these analyses, the aminoglycosides act as receptors, while RNA structures labeled with a benzofuranyluridine fluorophore act as analytes. Complete (100%) predictive ability for this RNA training set was achieved by incorporating two exhaustively guanidinylated aminoglycosides into the receptor library. The PCA was then externally validated using biologically relevant RNA constructs. In bulge-stem-loop constructs of HIV-1 transactivation response element (TAR) RNA, we achieved nucleotide-specific classification of two independent secondary structure motifs. Furthermore, examination of cheminformatic parameters and PCA loading factors revealed trends in aminoglycoside:RNA recognition, including the importance of shape-based discrimination, and suggested the potential for size and sequence discrimination within RNA structural motifs. These studies present a new approach to classifying RNA structure and provide direct evidence that RNA topology, in addition to sequence, is critical for the molecular recognition of RNA.

3' downstream inhibitory stem plays a crucial role in locking rpoS mRNA 5' untranslated region in a self-inhibitory state. Here, we used optical tweezers to study the unfolding/refolding of rpoS inhibitory stem in the absence and presence of Mg2+. We found adding Mg2+ decreased the free energy of the RNA junction without re-arranging its secondary structure, through confirming that this RNA formed a canonical RNA three-way junction. We suspected increased free energy might change the relative orientation of different stems of rpoS and confirmed this by small angle X-ray scattering. Such changed conformation may improve Hfq-bridged annealing between sRNA and rpoS RNA inhibitory stem. We established a convenient route to analyze the changes of RNA conformation and folding dynamics by combining optical tweezers with X-ray scattering methods. This route can be easily applied in the studies of other RNA structure and ligand-RNA.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs. In vivo probing of RNA structures with COMRADES yields insight into RNA folding of the ZIKA virus genome and its interaction with host RNAs.

Interactions between protein and RNA play a key role in many biological processes in the gene expression pathway. Those interactions are mediated through a variety of RNA-binding protein domains, among them the highly abundant RNA recognition motif (RRM). Here we studied protein-RNA complexes from different RNA binding domain families solved by NMR and x-ray crystallography. Characterizing the structural properties of the RNA at the binding interfaces revealed an unexpected number of nucleotides with unusual RNA conformations, specifically found in RNA-RRM complexes. Moreover, we observed that the RNA nucleotides that are directly involved in interactions with the RRM domains, via hydrogen bonds and hydrophobic contacts, are significantly enriched with unique RNA conformations. Further examination of the sequences binding the RRM domain showed a preference for G nucleotides in syn conformation to precede or to follow U nucleotides in the anti-conformation, and U nucleotides in C2' endo conformation to precede U and G nucleotides possessing the more common C3' endo conformation. These findings imply a possible mode of RNA recognition by the RRM domains which enables the recognition of a wide variety of different RNA sequences and shapes. Overall, this study suggests an additional way by which the RRM domain recognizes its RNA target, involving a conformational readout.

Comparison of kinetic and thermodynamic properties of IRP1 (iron regulatory protein1) binding to FRT (ferritin) and ACO2 (aconitase2) IRE-RNAs, with or without Mn2+, revealed differences specific to each IRE-RNA. Conserved among animal mRNAs, IRE-RNA structures are noncoding and bind Fe2+ to regulate biosynthesis rates of the encoded, iron homeostatic proteins. IRP1 protein binds IRE-RNA, inhibiting mRNA activity; Fe2+ decreases IRE-mRNA/IRP1 binding, increasing encoded protein synthesis. Here, we observed heat, 5 °C to 30 °C, increased IRP1 binding to IRE-RNA 4-fold (FRT IRE-RNA) or 3-fold (ACO2 IRE-RNA), which was enthalpy driven and entropy favorable. Mn2+ (50 µM, 25 °C) increased IRE-RNA/IRP1 binding (Kd) 12-fold (FRT IRE-RNA) or 6-fold (ACO2 IRE-RNA); enthalpic contributions decreased ~61% (FRT) or ~32% (ACO2), and entropic contributions increased ~39% (FRT) or ~68% (ACO2). IRE-RNA/IRP1 binding changed activation energies: FRT IRE-RNA 47.0 ± 2.5 kJ/mol, ACO2 IRE-RNA 35.0 ± 2.0 kJ/mol. Mn2+ (50 µM) decreased the activation energy of RNA-IRP1 binding for both IRE-RNAs. The observations suggest decreased RNA hydrogen bonding and changed RNA conformation upon IRP1 binding and illustrate how small, conserved, sequence differences among IRE-mRNAs selectively influence thermodynamic and kinetic selectivity of the protein/RNA interactions.

Internal ribosome entry site (IRES) elements are high-order RNA structures that promote internal initiation of translation to allow protein synthesis under situations that compromise the general cap-dependent translation mechanism. Picornavirus IRES elements are highly efficient elements with a modular RNA structure organization. Here we investigated the effect of Mg(2+) concentration on the local flexibility and solvent accessibility of the foot-and-mouth disease virus (FMDV) IRES element measured on the basis of selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reactivity and hydroxyl radical cleavage. We have found that Mg(2+) concentration affects the organization of discrete IRES regions, mainly the apical region of domain 3, the 10 nt loop of domain 4, and the pyrimidine tract of domain 5. In support of the effect of RNA structure on IRES activity, substitution or deletion mutants of the 10 nt loop of domain 4 impair internal initiation. In addition, divalent cations affect the binding of eIF4G, a eukaryotic initiation factor that is essential for IRES-dependent translation that interacts with domain 4. Binding of eIF4G is favored by the local RNA flexibility adopted at low Mg(2+) concentration, while eIF4B interacts with the IRES independently of the compactness of the RNA structure. Our study shows that the IRES element adopts a near-native structure in the absence of proteins, shedding light on the influence of Mg(2+) ions on the local flexibility and binding of eIF4G in a model IRES element.

Neomycin dimers synthesized using "click chemistry" with varying functionality and length in the linker region have been shown to be effective in targeting the HIV-1 TAR RNA region of the HIV virus. TAR (Transactivation Response) RNA region, a 59 base pair stem loop structure located at the 5'-end of all nascent viral transcripts interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Ethidium bromide displacement and FRET competition assays have revealed nanomolar binding affinity between neomycin dimers and wildtype TAR RNA while in case of neomycin, only a weak binding was detected. Here, NMR and FID-based comparisons reveal an extended binding interface for neomycin dimers involving the upper stem of the TAR RNA thereby offering an explanation for increased affinities. To further explore the potential of these modified aminosugars we have extended binding studies to include four TAR RNA mutants that display conformational differences with minimal sequence variation. The differences in binding between neomycin and neomycin dimers is characterized with TAR RNA mutants that include mutations to the bulge region, hairpin region, and both the bulge and hairpin regions. Our results demonstrate the effect of these mutations on neomycin binding and our results show that linker functionalities between dimeric units of neomycin can distinguish between the conformational differences of mutant TAR RNA structures.

RNA sequences having up to three consecutive internal amide linkages were synthesized and studied using UV and NMR spectroscopy. The amide modifications did not interfere with normal base-pairing and A-type RNA conformation. Three consecutive amides were well tolerated in the passenger strand of siRNA and caused little change in RNAi activity.

RNA molecules have highly versatile structures that can fold into myriad conformations, providing many potential pockets for binding small molecules. The increasing number of available RNA structures, in complex with proteins, small ligands and in free form, enables the design of new therapeutically useful RNA-binding ligands. Here we studied RNA ligand complexes from 10 RNA groups extracted from the protein data bank (PDB), including adaptive and non-adaptive complexes. We analyzed the chemical, physical, structural and conformational properties of binding pockets around the ligand. Comparing the properties of ligand-binding pockets to the properties of computed pockets extracted from all available RNA structures and RNA-protein interfaces, revealed that ligand-binding pockets, mainly the adaptive pockets, are characterized by unique properties, specifically enriched in rare conformations of the nucleobase and the sugar pucker. Further, we demonstrate that nucleotides possessing the rare conformations are preferentially involved in direct interactions with the ligand. Overall, based on our comprehensive analysis of RNA-ligand complexes, we suggest that the unique conformations adopted by RNA nucleotides play an important role in RNA recognition by small ligands. We term the recognition of a binding site by a ligand via the unique RNA conformations “RNA conformational readout.” We propose that “conformational readout” is a general way by which RNA binding pockets are recognized and selected from an ensemble of different RNA states.

Picornavirus RNAs initiate translation using a 5′ end-independent mechanism based on internal ribosome entry site (IRES) elements. Despite performing similar functions, IRES elements present in genetically distant RNAs differ in primary sequence, RNA secondary structure and trans-acting factors requirement. The lack of conserved features amongst IRESs represents obstacles for the understanding of the internal initiation process. RNA structure is tightly linked to picornavirus IRES activity, consistent with the conservation of RNA motifs. This study extends the functional relevance of evolutionary conserved motifs of foot-and-mouth disease virus (FMDV) IRES. SHAPE structural analysis of mutant IRESs revealed local changes in RNA flexibility indicating the existence of an interactive structure constrained by lateral bulges that maintain the RNA conformation necessary for IRES-mediated translation. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., BFU2011-25437; Fundación Ramón Areces

Experiments demonstrate that Mg(2+) is crucial for structure and function of RNA systems, yet the detailed molecular mechanism of Mg(2+) action on RNA is not well understood. We investigate the interplay between RNA and Mg(2+) at atomic resolution through ten 2-μs explicit solvent molecular dynamics simulations of the SAM-I riboswitch with varying ion concentrations. The structure, including three stemloops, is very stable on this time scale. Simulations reveal that outer-sphere coordinated Mg(2+) ions fluctuate on the same time scale as the RNA, and that their dynamics couple. Locally, Mg(2+) association affects RNA conformation through tertiary bridging interactions; globally, increasing Mg(2+) concentration slows RNA fluctuations. Outer-sphere Mg(2+) ions responsible for these effects account for 80% of Mg(2+) in our simulations. These ions are transiently bound to the RNA, maintaining interactions, but shuttled from site to site. Outer-sphere Mg(2+) are separated from the RNA by a single hydration shell, occupying a thin layer 3-5 Å from the RNA. Distribution functions reveal that outer-sphere Mg(2+) are positioned by electronegative atoms, hydration layers, and a preference for the major groove. Diffusion analysis suggests transient outer-sphere Mg(2+) dynamics are glassy. Since outer-sphere Mg(2+) ions account for most of the Mg(2+) in our simulations, these ions may change the paradigm of Mg(2+)-RNA interactions. Rather than a few inner-sphere ions anchoring the RNA structure surrounded by a continuum of diffuse ions, we observe a layer of outer-sphere coordinated Mg(2+) that is transiently bound but strongly coupled to the RNA.

Changes in RNA conformation can alter gene expression. The guanine quadruplex sequence (GQS) is an RNA motif that folds in the presence of K(+) ions. Changes in the conformation of this motif could be especially important in regulating gene expression in plants because intracellular K(+) concentrations often increase during drought stress. Little is known about the folding thermodynamics of RNA GQS. We show here that RNA GQS with tracts containing three G's [e.g., (GGGxx)(4)] have a modest dependence on the K(+) concentration, folding with no or even negative cooperativity (Hill coefficients ≤1), and are associated with populated folding intermediates. In contrast, GQS with tracts containing just two G's [e.g., (GGxx)(4)] have a steep dependence on the K(+) concentration and fold with positive cooperativity (Hill coefficients of 1.7-2.7) without significantly populating intermediate states. We postulate that in plants, the more stable G3 sequences are largely folded even under unstressed conditions, while the less stable G2 sequences fold only at the higher K(+) concentrations associated with cellular stress, wherein they respond sharply to changing K(+) concentrations. Given the binary nature of their folding, G2 sequences may find application in computation with DNA and in engineering of genetic circuits.

Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 × 2 bulged RNA and one 2 × 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 × 2 bulge (C65 and G66). The RNA WT construct was among the most stable (ΔG°37 = −7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A•G mismatch of the bulge decreases the stability of the construct ∼0.5–1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA–Mg2+ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/mol relative to 1 M salt. Formation of a single A+•C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+•C mismatch base pairs together contribute about 2 kcal/mol.

Various chemical modifications are currently being evaluated for improving the efficacy of short interfering RNA (siRNA) duplexes as antisense agents for gene silencing in vivo. Among the 2'-ribose modifications assessed to date, 2'deoxy-2'-fluoro-RNA (2'-F-RNA) has unique properties for RNA interference (RNAi) applications. Thus, 2'-F-modified nucleotides are well tolerated in the guide (antisense) and passenger (sense) siRNA strands and the corresponding duplexes lack immunostimulatory effects, enhance nuclease resistance and display improved efficacy in vitro and in vivo compared with unmodified siRNAs. To identify potential origins of the distinct behaviors of RNA and 2'-F-RNA we carried out thermodynamic and X-ray crystallographic analyses of fully and partially 2'-F-modified RNAs. Surprisingly, we found that the increased pairing affinity of 2'-F-RNA relative to RNA is not, as commonly assumed, the result of a favorable entropic contribution ('conformational preorganization'), but instead primarily based on enthalpy. Crystal structures at high resolution and osmotic stress demonstrate that the 2'-F-RNA duplex is less hydrated than the RNA duplex. The enthalpy-driven, higher stability of the former hints at the possibility that the 2'-substituent, in addition to its important function in sculpting RNA conformation, plays an underappreciated role in modulating Watson-Crick base pairing strength and potentially π-π stacking interactions.

The hepatitis C virus internal ribosome entry site (IRES) element contains a three-way junction that is important in the overall RNA conformation, and for its role in the internal initiation of translation. The junction also illustrates some important conformational principles in the folding of three-way helical junctions. It is formally a 3HS4 junction, with the possibility of two alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would form 2HS1HS3 and 2HS2HS2 junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer (FRET) studies show that the junction is induced to fold by the presence of Mg2+ ions in low micromolar concentrations, and suggest that the structure adopted is based on coaxial stacking of the two helices that do not terminate in a hairpin loop (i.e., helix IIId). Single-molecule FRET studies confirm this conclusion, and indicate that there is no minor conformer present based on an alternative choice of helical stacking partners. Moreover, analysis of single-molecule FRET data at an 8-msec resolution failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique and stable fold.

Proteins play diverse and critical roles in cellular ribonucleoproteins (RNPs) including promoting formation of and stabilizing active RNA conformations. Yet, the conformational changes required to convert large RNAs into an active RNPs have proven difficult to characterize fully. Here we use high-resolution approaches to monitor both local nucleotide flexibility and solvent accessibility for nearly all nucleotides in the bI3 group I intron RNP in four assembly states: the free RNA, maturase-bound RNA, Mrs1-bound RNA, and the complete six-component holocomplex. The free RNA is misfolded relative to the secondary structure required for splicing. The maturase and Mrs1 proteins each stabilized long-range tertiary interactions but neither protein alone induced folding into the functional secondary structure. In contrast, simultaneous binding by both proteins results in large secondary structure rearrangements in the RNA and yielded the catalytically active group I intron structure. Secondary and tertiary folding of the RNA component of the bI3 RNP are thus not independent: RNA folding is strongly non-hierarchical. These results emphasize that protein-mediated stabilization of RNA tertiary interactions functions to pull the secondary structure into an energetically disfavored, but functional, conformation and emphasize a new role for facilitator proteins in RNP assembly.

Microtubule-based mRNA transport is widely used to restrict protein expression to specific regions in the cell and has important roles in defining cell polarity and axis determination as well as in neuronal function. However, the structural basis of recognition of cis-acting mRNA localization signals by motor complexes is poorly understood. We have used NMR spectroscopy to describe the first tertiary structure to our knowledge of an RNA element responsible for mRNA transport. The Drosophila melanogaster fs(1)K10 signal, which mediates transport by the dynein motor, forms a stem loop with two double-stranded RNA helices adopting an unusual A'-form conformation with widened major grooves reminiscent of those in B-form DNA. Structure determination of four mutant RNAs and extensive functional assays in Drosophila embryos indicate that the two spatially registered A'-form helices represent critical recognition sites for the transport machinery. Our study provides insights into the basis for RNA cargo recognition and reveals a key biological function encoded by A'-form RNA conformation.

Interaction of the protoberberine alkaloids berberine, palmatine, and coralyne with the two double-stranded RNA homopolymers of cytidine-guanosine (CG) and inosine-cytidine (IC) sequences in the protonated conformation was investigated using various biophysical techniques. All the three alkaloids bound polyC(+)G in a cooperative way. The binding of coralyne to both the polyribonucleotides was stronger than that of berberine and palmatine. Evidence for the intercalative binding of coralyne was revealed from fluorescence quenching studies. Isothermal titration calorimetry results suggested that the binding of berberine to both the polymers and palmatine to polyIC(+) was very weak while that of palmatine and coralyne to polyC(+)G and polyIC(+) was predominantly entropy driven. Circular dichroic results provided evidence for the perturbation of the RNA conformation with the bound coralyne in a more deeply intercalated position compared to berberine and palmatine as revealed by induced circular dichroism peaks. Taken together, the present study suggests that planarity of coralyne results in a more favorable and stronger binding to the double-stranded RNA conformations compared to berberine and palmatine that may potentiate its use in RNA-targeted drug design.

The ribosome is a complex macromolecular machine responsible for protein synthesis in the cell. It consists of two subunits, each of which contains both RNA and protein components. Ribosome assembly is subject to intricate regulatory control and is aided by a multitude of assembly factors in vivo, but can also be carried out in vitro. The details of the assembly process remain unknown even in the face of atomic structures of the entire ribosome and after more than three decades of research. Some of the earliest research on ribosome assembly produced the Nomura assembly map of the small subunit, revealing a hierarchy of protein binding dependencies for the 20 proteins involved and suggesting the possibility of a single intermediate. Recent work using a combination of RNA footprinting and pulse-chase quantitative mass spectrometry paints a picture of small subunit assembly as a dynamic and varied landscape, with sequential and hierarchical RNA folding and protein binding events finally converging on complete subunits. Proteins generally lock tightly into place in a 5′ to 3′ direction along the ribosomal RNA, stabilizing transient RNA conformations, while RNA folding and the early stages of protein binding are initiated from multiple locations along the length of the RNA.

Deoxyribonuclease I (DNase I) binds right-handed DNA duplex via a minor groove and the backbone phosphate group with no contact to the major groove. It hydrolyses double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions, in the presence of divalent Mg and Ca cations. Even though DNase-RNA interaction was observed, less is known about the protein-RNA binding mode and the effect of such complexation on both protein and RNA conformations. The aim of this study was to examine the effects of DNase I-tRNA interaction on tRNA and protein conformations. The interaction of DNase I with tRNA is monitored under physiological conditions, in the absence of Mg2+, using constant DNA concentration of 12.5 mM (phosphate) and various protein contents (10 microM to 250 microM). FTIR, UV-visible, and CD spectroscopic methods were used to analyze the protein binding mode, the binding constant, and the effects of polynucleotide-enzyme interaction on both tRNA and protein conformations. Spectroscopic evidence showed major DNase-PO2 and minor groove interactions with overall binding constant of K = 2.1 (+/-0.7) x 10(4) M(-1). The DNase I-tRNA interaction alters protein secondary structure with major reduction of the alpha-helix, and increases the random coil, beta-anti and turn structures, while tRNA remains in the A-conformation. No digestion of tRNA by DNase I was observed in the protein-tRNA complexes.

Steric-block ON analogues are efficient inhibitors of RNA-protein interaction and therefore have potential to probe RNA sequences for putative protein binding sites and to investigate mechanisms of protein binding. The packaging process of HIV-1 is highly specific involving an interaction between the Gag protein and a conserved sequence that is only present on genomic viral RNA. Using oligonucleotide probes we have confirmed that the terminal purine loop is the major Gag binding site on SL3 and that a secondary Gag binding site exists at an internal purine bulge. We also demonstrate direct binding of oligonucleotide to their binding sites and confirm this interaction does not alter global RNA conformation, making them highly specific, nondisruptive probes of RNA protein interactions.

BglG/LicT-like proteins are transcriptional antiterminators that prevent termination of transcription at intrinsic terminators by binding to ribonucleic antiterminator (RAT) sites and stabilizing an RNA conformation which is mutually exclusive with the terminator structure. The known RAT sites, which are located in intergenic regions of sugar utilization operons, show low sequence conservation but significant structural analogy. To assess the prevalence of RATs in bacterial genomes, we employed bioinformatic tools that describe RNA motifs based on both sequence and structural constraints. Using descriptors with different stringency, we searched the genomes of Escherichiacoli K12, uropathogenic E. coli and Bacillus subtilis for putative RATs. Our search identified all known RATs and additional putative RAT elements. Surprisingly, most putative RATs do not overlap an intrinsic terminator and many reside within open reading frames (ORFs). The ability of one of the putative RATs, which is located within an antiterminator-encoding ORF and does not overlap a terminator, to bind to its cognate antiterminator protein in vitro and in vivo was confirmed experimentally. Our results suggest that the capacity of RAT elements has been exploited during evolution to mediate activities other than antitermination, for example control of transcription elongation or of RNA stability.

Many of the spliceosomal small nuclear RNAs (snRNAs) undergo large-scale structural rearrangements during splicing. This is exemplified by the U6 snRNA which can exist in isolation in the U6 small nuclear ribonucleoprotein (snRNP); basepaired with U4 in the U4/U6 di-snRNP, U4/U6.U5 tri-snRNP, and spliceosomal B complex; or basepaired with the pre-mRNA intron and the U2 snRNA in the catalytic spliceosome. Through genetic evidence and structure probing from the Brow, Guthrie, and Luhrmann labs, it was previously proposed that the U4/U6 di-snRNA consisted of a three-helical junction core domain flanked by single stranded regions. Using single-molecule Forster resonance energy transfer (smFRET), we discovered a dynamic equilibrium between RNA conformations formed by the flanking single stranded regions. Our smFRET data is consistent with the presence of two mutually exclusive structures: (1) a U4/U6 di-snRNA containing both basepaired U4/U6 and the U6 telestem and (2) a U4/U6 di-snRNA with extended basepairin...

We analysed conformational changes of yeast tRNA(Phe) induced by high hydrostatic pressure (HHP) measured by Fourier-transform infrared (FTIR) and fluorescence spectroscopies. High pressure influences RNA conformation without other cofactors, such as metal ions and salts. FTIR spectra of yeast tRNA(Phe) recorded at high hydrostatic pressure up to 13 kbar with and without magnesium ions showed a shift of the bands towards higher frequencies. That blue shift is due to an increase a higher energy of bonds as a result of shortening of hydrogen bonds followed by dehydration of tRNA. The fluorescence spectra of Y-base tRNA(Phe) at high pressure up to 3 kbar showed a decrease of the intensity band at 430 nm as a consequence of conformational rearrangement of the anticodon loop leading to exposure of Y-base side chain to the solution. We suggest that structural transition of nucleic acids is driven by the changes of water structure from tetrahedral to a cubic-like geometry induced by high pressure and, in consequence, due to economy of hydration.

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25 degrees C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding.

The targeting of RNA for the design of novel anti-viral compounds represents an area of vast potential. We have used NMR and computational methods to model the interaction of a series of synthetic inhibitors of the in vitro RNA binding activities of a peptide derived from the transcriptional activator protein, Tat, from human immunodeficiency virus type 1. Inhibition has been measured through the monitering of fluorescence resonance energy transfer between fluorescently labeled peptide and RNA components. A series of compounds containing a bi-aryl heterocycle as one of the three substituents on a benzylic scaffold, induce a novel, inactive TAR conformation by stacking between base-pairs at the site of a three-base bulge within TAR. The development of this series resulted in an enhancement in potency (with Ki < 100 nM in an in vitro assay) and the removal of problematic guanidinium moieties. Ligands from this series can act as inhibitors of Tat-induced transcription in a cell-free system. This study validates the drug design strategy of using a ligand to target the RNA receptor in a non-functional conformation.

Dipeptide-conjugated nucleosides were efficiently synthesized from the intermediates of 3′-amino-3′-deoxy-nucleosides by using the solid-phase synthetic strategy with HOBt/HBTU (1-hydroxy-1H-benzotriazole/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoroborate) as the coupling reagents (Schemes 1–3). CD Spectra and thermal melting studies showed that the synthesized hydrophobic dipeptidethymidine and uridine derivatives 8a–8d, 13a–d, and 18 had a mild affinity with the polyA⋅polyU duplex and could induce the change of RNA conformation. The results also implied that the interaction of conjugates with RNA might be related to the sugar pucker conformation of the nucleoside.

A simple method for rapid structure screening for RNA by NMR is proposed. Target RNA is transcribed in a NMR tube and its spectra are measured without purification. The proposed method, in NMR tube transcription or INTT, was applied for three RNAs for which NMR spectra have been measured by using the conventionally purified samples. By the real-time measuring, increase of imino proton signals and decrease of NTP signals can be observed. It was confirmed that INTT spectra are in general similar to those obtained by the conventional method. INTT can be used for the first-step screening of RNA folding.

The signal recognition particle (SRP) targets proteins to the endoplasmic reticulum in eukaryotes or to the inner membrane in prokaryotes by binding to hydrophobic signal sequences. Signal peptide recognition occurs within the highly conserved RNA-protein core of the SRP, underscoring the importance of this complex in SRP function. Structural analysis of the RNA and protein components of the prokaryotic SRP in the free and bound states revealed that the RNA undergoes a significant conformational change upon protein binding involving the uptake of several monovalent and divalent cations. To investigate the role of these metal ions in formation of the functional SRP complex, we used binding affinity assays and X-ray crystallography to analyze the specificity and energetic contributions of mono- and divalent metal ions bound in the RNA. Our results demonstrate that several metal ion binding sites important for RNA conformation can accommodate chemically distinct ions, often without affecting the structure of the complex. Thus, while these metal ions are highly ordered and essential for the formation and stability of the SRP complex, they behave like nonspecific metal ions.

In cells, RNAs likely adopt numerous intermediate conformations prior to formation of functional RNA-protein complexes. We used single-nucleotide resolution selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to probe the structure of Escherichia coli 16S rRNA in healthy growing bacteria. SHAPE-directed modeling indicated that the predominant steady-state RNA conformational ensemble in dividing cells had a base-paired structure different from that expected on the basis of comparative sequence analysis and high-resolution studies of the 30S ribosomal subunit. We identified the major cause of these differences by stopping ongoing in-cell transcription (in essence, an in-cell RNA structure pulse-chase experiment) which caused the RNA to chase into a structure that closely resembled the expected one. Most helices that formed alternate RNA conformations under growth conditions interact directly with tertiary-binding ribosomal proteins and form a C-shape that surrounds the mRNA channel and decoding site. These in-cell experiments lead to a model in which ribosome assembly factors function as molecular struts to preorganize this intermediate and emphasize that the final stages of ribonucleoprotein assembly involve extensive protein-facilitated RNA conformational changes.

Pseudouridine is present in ribosomal RNA, transfer RNA, tmRNA, and small nuclear and nucleolar RNAs. All are structured molecules. Pseudouridine is made by enzyme-catalyzed isomerization of specifically selected U residues after the polynucleotide chain is made. No energy input is required. Pseudouridine formation creates a hydrogen bond donor at the equivalent of uridine C-5. Therefore, a major role of pseudouridine may be to strengthen particular RNA conformations and/or RNA-RNA interactions because of this extra H-bond capability. Understanding the role of pseudouridine critically depends on knowledge of their location and number in RNA. The mapping method described here has greatly simplified this task and made it possible to survey many organisms. Procedures are described for mapping pseudouridines in large RNAs like ribosomal RNA and in small RNAs like tRNA. The method involves carbodiimide adduct formation with U, G, and pseudouridine followed by mild alkali to remove the adduct from U and G but not from the N-3 of pseudouridine. This results in attenuation of primed reverse transcription resulting in a stop band one residue 3' to the pseudouridine on sequencing gels. Use of primers means that purified RNAs are not needed, only knowledge of its primary sequence, but limit the sequence scanned to 30-40 residues from the 3' end. A poly(A) tailing procedure is described that allows extension of the method to within a few nucleotides of the 3' terminus.

Virtually all RNA-mediated reactions require transitions among alternative RNA conformations. The complexity of biological reactions can obscure specific conformational changes in vivo and important features of the intracellular environment are difficult to reproduce in vitro. However, simple RNA self-cleavage and ligation reactions offer a unique opportunity to measure the kinetics and equilibria of specific RNA conformational transitions directly in living cells. Hairpin ribozymes that incorporate the natural four-way helical junction self-cleave rapidly in vivo, but only when cleavage products dissociate rapidly. Cleavage rates fall when cleavage products remain bound in stable base-paired helices, providing evidence that bound products undergo re-ligation. These results provide the first detailed kinetic description of an intracellular ribozyme reaction that includes cleavage, ligation and product dissociation rates. Kinetic and equilibrium parameters measured in vivo correspond well, but not perfectly, with values measured for the same reactions in vitro under conditions that approximate an intracellular ionic environment.

A new type of structural compensation between the lengths of two perpendicularly oriented RNA double helices was found in the archaeal selenocysteine tRNA from Methanococcus jannascii. This tRNA contains only four base-pairs in the T-stem, one base-pair less than in all other cytosolic tRNAs. Our analysis shows that such a T-stem in an otherwise normal tRNA cannot guarantee the formation of the normal interactions between the D and T-loops. The absence of these interactions would affect the juxtaposition of the two tRNA helical domains, potentially damaging the tRNA function. In addition to the short T-stem, this tRNA possesses another unprecedented feature, a very long D-stem consisting of seven base-pairs. Taken as such, a seven base-pair D-stem will also disrupt the normal interaction between the D and T-loops. On the other hand, the presence of the universal nucleotides in both the D and T-loops suggests that these loops probably interact with each other in the same way as in other tRNAs. Here, we demonstrate that the short T-stem and the long D-stem can naturally compensate each other, thus providing the normal D/T interactions. Molecular modeling has helped suggest a detailed scheme of mutual compensation between these two unique structural aspects of the archaeal selenocysteine tRNA. In the light of this analysis, other structural and functional characteristics of the selenocysteine tRNAs are discussed.

Evidence is presented for the participation of the P loop (nucleotides G2250-C2254) of 23 S rRNA in establishing the tertiary structure of the peptidyl transferase center. Single base substitutions were introduced into the P loop, which participates in peptide bond formation through direct interaction with the CCA end of P site-bound tRNA. These mutations altered the pattern of reactivity of RNA to chemical probes in a structural subdomain encompassing the P loop and extending roughly from G2238 to A2433. Most of the effects on chemical modification in the P loop subdomain occurred near sites of tertiary interactions inferred from comparative sequence analysis, indicating that these mutations perturb the tertiary structure of this region of RNA. Changes in chemical modification were also seen in a subdomain composed of the 2530 loop (nucleotides G2529-A2534) and the A loop (nucleotides U2552-C2556), the latter a site of interaction with the CCA end of A site-bound tRNA. Mutations in the P loop induced effects on chemical modification that were commensurate with the severity of their characterized functional defects in peptide bond formation, tRNA binding and translational fidelity. These results indicate that, in addition to its direct role in peptide bond formation, the P loop contributes to the tertiary structure of the peptidyl transferase center and influences the conformation of both the acceptor and peptidyl tRNA binding sites.

In keeping with the known role of polyamines as counterions in RNA folding, we have found that concentrations of spermine as low as 1 microM facilitate first-order Cis cleavage and decrease the concentration of magnesium required for optimal cleavage of the VS ribozyme. Surprisingly, under certain experimental conditions, cleavage reactions at concentrations of spermine above about 20 microM were not first-order. At 100 microM spermine, about half of the RNA cleaved in an initial very fast burst, k >/= 5 min-1, about 100-fold faster than under our previously optimized conditions; the remainder of the RNA cleaved more slowly. The extent of the burst and the initial rate of cleavage were proportional to RNA concentration, suggesting that the fast phase was due to intermolecular trans cleavage involving two or more RNAs. This inference of trans cleavage was confirmed by demonstrating spermine-dependent trans cleavage of particular combinations of mutant RNAs that were each incapable of cis cleavage. The experimental conditions required to switch the VS ribozyme into trans cleavage mode are quite stringent. The RNA must be preincubated with an adequate concentration of spermine at very low ionic strength near neutral pH. Concentrations of buffers and salts typically used in in vitro studies of ribozymes, including those used in our previous characterization of the VS ribozyme, are sufficiently high that they prevent or reverse the trans cleaving RNA conformation. The ability to switch cleavage modes from cis to trans provides an experimental system to study different active conformations of VS RNA, as well as to investigate the functional consequences of polyamine-RNA interactions.

Mg2+ has been shown to modulate the function of riboswitches by facilitating the ligand-riboswitch interactions. The btuB riboswitch from Escherichia coli undergoes a conformational change upon binding to its ligand, coenzyme B12 (adenosyl-cobalamine, AdoCbl), and down-regulates the expression of the B12 transporter protein BtuB in order to control the cellular levels of AdoCbl. Here, we discuss the structural folding attained by the btuB riboswitch from E. coli in response to Mg2+ and how it affects the ligand binding competent conformation of the RNA. The btuB riboswitch notably adopts different conformational states depending upon the concentration of Mg2+. With the help of in-line probing, we show the existence of at least two specific conformations, one being achieved in the complete absence of Mg2+ (or low Mg2+ concentration) and the other appearing above ∼0.5 mM Mg2+. Distinct regions of the riboswitch exhibit different dissociation constants toward Mg2+, indicating a stepwise folding of the btuB RNA. Increasing the Mg2+ concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg2+ defines the binding competent conformation of the btuB riboswitch which can productively interact with the ligand, coenzyme B12, and switch the RNA conformation. Moreover, raising the Mg2+ concentration enhances the ratio of switched RNA in the presence of AdoCbl. The lack of a AdoCbl-induced conformational switch experienced by the btuB riboswitch in the absence of Mg2+ indicates a crucial role played by Mg2+ for defining an active conformation of the riboswitch.

Gene expression from the human immunodeficiency virus 1 (HIV-1) is greatly enhanced by binding of the virally encoded Tat protein to a 59-base RNA stem-loop structure, the Transactivation Responsive Element (TAR), located at the 5'-termini of all viral transcripts. This interaction was investigated in vitro using 32P-labelled TAR and highly purified Tat in which cysteine residues were blocked by sulpitolysis (S-Tat). It is shown that specific complex formation between S-Tat and TAR can occur in the presence of urea, with urea concentrations between 5 and 6 M causing an approximately two-fold increase in the level of binding. Two conditions favoring RNA secondary structure, low temperature (0 degree C) and the presence of divalent cations (Mg2+), diminished the level of specific binding. These observations suggest that the presence of mild denaturants promoted macromolecular refolding or rearrangement in a manner that increased the number of molecules available for binding, and present a general method for studying protein/RNA interactions where analysis has been obstructed by improper protein or RNA conformation.

Diethylstilbestrol (DES), a synthetic estrogen, is known to be a carcinogen in human and in animals. This study was designed to examine the interaction of DES with yeast RNA in aqueous solution at physiological pH with drug/RNA-phosphate (P) molar ratios of 1/80, 1/40, 1/20, 1/10, 1/4, and 1/2. Fourier transform infrared (FTIR) difference spectroscopy was used to determine the drug binding mode, the binding constant, the sequence selectivity, and RNA secondary structure in the RNA.DES complexes. Spectroscopic evidence showed that at low drug concentration (1/80 and 1/40), DES is intercalating through both Gua-Cyt and Ade-Urd base pairs with minor interaction with the backbone PO2 group (external binding). The calculated binding constant of K approximately 8.5 x 10(4) M-1 at a drug concentration of 3.12 x 10(-4) M shows that DES is a weaker intercalator than those of the methylene blue, acridine orange, and ethidium bromide. At high drug content (r1/40, where r represents the DES/RNA-phosphate molar ratio), a partial helix destabilization occurs with no alteration of RNA conformation upon drug complexation. However, a comparison with DNA.DES complexes showed that drug intercalation causes major reduction of the B-DNA structure in favor of A-DNA with no participation of the backbone PO2 group in the DES. DNA complexation.

A single arginine residue within the RNA-binding domain of the human immunodeficiency virus Tat protein makes a critical sequence-specific contact to TAR RNA. Arginine as the free amino acid also binds specifically to TAR and induces a change in RNA conformation similar to that induced by Tat peptides. NMR and biochemical studies have suggested that the arginine-binding site is stabilized by a base triple interaction between a bulged U and an A x U base pair in the adjacent stem. In this study, we have used chemical modification and mutagenesis experiments to examine the relative contributions of the Watson-Crick and Hoogsteen base-pairing partners of the proposed U-A x U base triple. We show that the Hoogsteen interaction is critical for arginine binding whereas the Watson-Crick interaction can be eliminated or replaced by other base-base interactions. The results are consistent with biochemical studies of the Tat-TAR interaction and support the base triple model for the structure of TAR.