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Competing Interests: Disclosures The authors have no conflicts of interest to disclose.

Direct RNA nanopore sequencing reveals changes in gene expression, polyadenylation, splicing, m6A methylation, and pseudouridylation in response to influenza virus exposure in primary human bronchial epithelial cells. This study focuses on the epitranscriptomic profile of genes in the host immune response. In addition to polyadenylated noncoding RNA, we purified and sequenced nonpolyadenylated noncoding RNA and observed changes in expression, N6-methyl-adenosine (m6A), and pseudouridylation (Ψ) in these novel RNA. Two recently discovered lincRNA with roles in immune response, Chaserr and LEADR , became highly methylated in response to influenza exposure. Several H/ACA type snoRNAs that guide pseudouridylation are decreased in expression in response to influenza, and there is a corresponding decrease in the pseudouridylation of two novel lncRNA. Thus, novel epitranscriptomic changes revealed by direct RNA sequencing with nanopore technology provides unique insights into the host epitranscriptomic changes in epithelial gene networks that respond to influenza virus infection.

Competing Interests: Declaration of interests The author declares no competing interests.

Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated 5'GGUC3' paired to 3'CUGG5' separately from other 5'GU3'/3'UG5' stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37°C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and assemble new virus particles. These structurally dynamic RNA shapeshifters present a challenging RNA folding problem, because the RNA sequence adopts multiple structures and may sometimes contain regions of partial disorder. Recent advances in high resolution asymmetric cryoelectron microscopy and chemical probing provide new ways to probe the degree of structure and disorder, and have identified more than one conformation in dynamic equilibrium in viral RNA. Chemical probing and the Detection of RNA Folding Ensembles using Expectation Maximization (DREEM) algorithm has been applied to studies of the dynamic equilibrium conformations in HIV RNA in vitro, in virio, and in vivo. This new type of data provides insight into important questions about virus assembly mechanisms and the fundamental physical forces driving virus particle assembly.

The revolution in sequencing technology demands new tools to interpret the genetic code. As in vivo transcriptome-wide chemical probing techniques advance, new challenges emerge in the RNA folding problem. The emphasis on one sequence folding into a single minimum free energy structure is fading as a new focus develops on generating RNA structural ensembles and identifying functional structural features in ensembles. This review describes an efficient combinatorially complete method and three free energy minimization approaches to predicting RNA structures with more than one functional fold, as well as two methods for analysis of a thermodynamics-based Boltzmann ensemble of structures. The review then highlights two examples of viral RNA 3'-UTR regions that fold into more than one conformation and have been characterized by single molecule fluorescence energy resonance transfer or NMR spectroscopy. These examples highlight the different approaches and challenges in predicting structure and function from sequence for RNA with multiple biological roles and folds. More well-defined examples and new metrics for measuring differences in RNA structures will guide future improvements in prediction of RNA structure and function from sequence., (© 2018 Schroeder; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

GU base pairs are important RNA structural motifs and often close loops. Accurate prediction of RNA structures relies upon understanding the interactions determining structure. The thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs are not well understood. Here, several self-complementary oligonucleotide sequences expected to form duplexes with 2 × 2 nucleotide internal loops closed by GU pairs were investigated. Surprisingly, nuclear magnetic resonance revealed that many of the sequences exist in equilibrium between hairpin and duplex conformations. This equilibrium is not observed with loops closed by Watson-Crick pairs. To measure the thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs, non-self-complementary sequences that preclude formation of hairpins were designed. The measured thermodynamics indicate that some internal loops closed by GU pairs are unusually unstable. This instability accounts for the observed equilibria between duplex and hairpin conformations. Moreover, it suggests that future three-dimensional structures of loops closed by GU pairs may reveal interactions that unexpectedly destabilize folding.

Background: The sequence of nucleotides in an RNA determines the possible base pairs for an RNA fold and thus also determines the overall shape and function of an RNA. The Swellix program presented here combines a helix abstraction with a combinatorial approach to the RNA folding problem in order to compute all possible non-pseudoknotted RNA structures for RNA sequences. The Swellix program builds on the Crumple program and can include experimental constraints on global RNA structures such as the minimum number and lengths of helices from crystallography, cryoelectron microscopy, or in vivo crosslinking and chemical probing methods., Results: The conceptual advance in Swellix is to count helices and generate all possible combinations of helices rather than counting and combining base pairs. Swellix bundles similar helices and includes improvements in memory use and efficient parallelization. Biological applications of Swellix are demonstrated by computing the reduction in conformational space and entropy due to naturally modified nucleotides in tRNA sequences and by motif searches in Human Endogenous Retroviral (HERV) RNA sequences. The Swellix motif search reveals occurrences of protein and drug binding motifs in the HERV RNA ensemble that do not occur in minimum free energy or centroid predicted structures., Conclusions: Swellix presents significant improvements over Crumple in terms of efficiency and memory use. The efficient parallelization of Swellix enables the computation of sequences as long as 418 nucleotides with sufficient experimental constraints. Thus, Swellix provides a practical alternative to free energy minimization tools when multiple structures, kinetically determined structures, or complex RNA-RNA and RNA-protein interactions are present in an RNA folding problem.

Accurate thermodynamic parameters improve RNA structure predictions and thus accelerate understanding of RNA function and the identification of RNA drug binding sites. Many viral RNA structures, such as internal ribosome entry sites, have internal loops and bulges that are potential drug target sites. Current models used to predict internal loops are biased toward small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with >6 nucleotides (nt) that occur frequently in viral RNA. This article presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Uracil and protonated cytosine base pairs stabilize asymmetric internal loops. Accurate prediction rules are presented that account for all thermodynamic measurements of RNA asymmetric internal loops. New loop initiation terms for loops with >6 nt are presented that do not follow previous assumptions that increasing asymmetry destabilizes loops. Since the last 2004 update, 126 new loops with asymmetry or sizes greater than 2 × 2 have been measured. These new measurements significantly deepen and diversify the thermodynamic database for RNA. These results will help better predict internal loops that are larger, pyrimidine-rich, and occur within viral structures such as internal ribosome entry sites., (© 2017 Phan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The thermodynamic stabilities of four natural prohead or packaging RNA (pRNA) three-way junction (3WJ) nanomotifs and seven phi29 pRNA 3WJ deletion mutant nanomotifs were investigated using UV optical melting on a three-component RNA system. Our data reveal that some pRNA 3WJs are more stable than the phi29 pRNA 3WJ. The stability of the 3WJ contributes to the unique self-assembly properties of pRNA. Thus, ultrastable pRNA 3WJ motifs suggest new scaffolds for pRNA-based nanotechnology. We present data demonstrating that pRNA 3WJs differentially respond to the presence of metal ions. A comparison of our data with free energies predicted by currently available RNA secondary structure prediction programs shows that these programs do not accurately predict multibranch loop stabilities. These results will expand the existing parameters used for RNA secondary structure prediction from sequence in order to better inform RNA structure-function hypotheses and guide the rational design of functional RNA supramolecular assemblies., (© 2017 Hill and Schroeder; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Metal ions are critical for RNA structure and enzymatic activity. We present the structure of an asymmetric RNA loop that binds metal ions and has an essential function in a bacteriophage packaging motor. Prohead RNA is a noncoding RNA that is required for genome packaging activity in phi29-like bacteriophage. The loops in GA1 and phi29 bacteriophage share a conserved adenine that forms a base triple, although the structural context for the base triple differs. NMR relaxation studies and femtosecond time-resolved fluorescence spectroscopy reveal the dynamic behavior of the loop in the metal ion bound and unbound forms. The mechanism of metal ion binding appears to be an induced conformational change between two dynamic ensembles rather than a conformational capture mechanism. These results provide experimental benchmarks for computational models of RNA-metal ion interactions.

Prohead RNA (pRNA) is an essential component of the powerful Φ29-like bacteriophage DNA packaging motor. However, the specific role of this unique RNA in the Φ29 packaging motor remains unknown. This review examines pRNA as a noncoding RNA of novel structure and function. In order to highlight the reasons for exploring the structure and function of pRNA, we (1) provide an overview of Φ29-like bacteriophage and the Φ29 DNA packaging motor, including putative motor mechanisms and structures of its component parts; (2) discuss pRNA structure and possible roles for pRNA in the Φ29 packaging motor; (3) summarize pRNA self-assembly; and (4) describe the prospective therapeutic applications of pRNA. Many questions remain to be answered in order to connect what is currently known about pRNA structure to its novel function in the Φ29 packaging motor. The knowledge gained from studying the structure, function, and sequence variation in pRNA will help develop tools to better navigate the conformational landscapes of RNA. WIREs RNA 2016, 7:428-437. doi: 10.1002/wrna.1330 For further resources related to this article, please visit the WIREs website., (© 2016 The Authors. WIREs RNA published by Wiley Periodicals, Inc.)

The folding landscape for an RNA sequence contains many diverse structures and motifs, which are often sampled rather than completely explored. Today's supercomputers make the complete enumeration of all possible folds for an RNA and a detailed description of the RNA folding landscape a more feasible task. This chapter provides protocols for using the Crumple folding algorithm, an efficient tool to generate all possible non-pseudoknotted folds for an RNA sequence. Crumple in conjunction with Sliding Windows and Assembly can incorporate experimental constraints on the global features of an RNA, such as the minimum number and lengths of helices, which may be determined by crystallography or cryo-electron microscopy. This complete enumeration method is independent of free-energy minimization and allows the user to incorporate experimental data such as chemical probing, SELEX data on RNA-protein binding motifs, and phylogenetic covariation.

Consecutive G·U base pairs inside RNA helices can be destabilizing, while those at the ends of helices are thermodynamically stabilizing. To determine if this paradox could be explained by differences in base stacking, we determined the high-resolution (1.32 Å) crystal structure of (5'-GGUGGCUGUU-3')2 and studied three sequences with four consecutive terminal G·U pairs by NMR spectroscopy. In the crystal structure of (5'-GGUGGCUGUU-3')2, the helix is overwound but retains the overall features of A-form RNA. The penultimate base steps at each end of the helix have high base overlap and contribute to the unexpectedly favorable energetic contribution for the 5'-GU-3'/3'-UG-5' motif in this helix position. The balance of base stacking and helical twist contributes to the positional dependence of G·U pair stabilities. The energetic stabilities and similarity to A-form RNA helices suggest that consecutive G·U pairs would be recognized by RNA helix binding proteins, such as Dicer and Ago. Thus, these results will aid future searches for target sites of small RNAs in gene regulation.

We present new modifications to the Wuchty algorithm in order to better define and explore possible conformations for an RNA sequence. The new features, including parallelization, energy-independent lonely pair constraints, context-dependent chemical probing constraints, helix filters, and optional multibranch loops, provide useful tools for exploring the landscape of RNA folding. Chemical probing alone may not necessarily define a single unique structure. The helix filters and optional multibranch loops are global constraints on RNA structure that are an especially useful tool for generating models of encapsidated viral RNA for which cryoelectron microscopy or crystallography data may be available. The computations generate a combinatorially complete set of structures near a free energy minimum and thus provide data on the density and diversity of structures near the bottom of a folding funnel for an RNA sequence. The conformational landscapes for some RNA sequences may resemble a low, wide basin rather than a steep funnel that converges to a single structure.

Viral RNA structure prediction is a valuable tool for development of drugs against viral disease. This work discusses different approaches to predicting encapsidated viral RNA and highlights satellite tobacco mosaic virus (STMV) RNA as a model system with excellent crystallography data. Fundamentally important issues for debate include thermodynamic versus kinetic control of virus assembly and the possible consequences of quasi-species in the primary structure on RNA secondary structure prediction of a single structure or an ensemble of structures. Multiple computational tools and chemical reagents are now available for improved viral RNA structure prediction. Two different predicted structures for encapsidated STMV RNA result from differences in three main areas: a different approach and philosophy to studying encapsidated viral RNA, an emphasis on different RNA motifs, and technical differences in computational methods and chemical reagents. The experiments with traditional chemical probing and SHAPE reagents are compared in terms of chemistry, results, and interpretation for STMV RNA as well as other RNA protein assemblies, such as the 5'UTR of HIV and the ribosome. This discussion of the challenges of viral RNA structure prediction will lead to new experiments and improved future predictions for viral RNA.

Consecutive terminal mismatches add thermodynamic stability to RNA duplexes and occur frequently in microRNA-mRNA interactions. Accurate thermodynamic stabilities of consecutive terminal mismatches contribute to the development of specific, high-affinity siRNA therapeutics. Consecutive terminal adenosine mismatches (TAMS) are studied at different salt concentrations, with polyethylene glycol cosolutes, and with locked nucleic acid (LNA) substitutions. These measurements provide benchmarks for the application of thermodynamic predictions to different physiological or therapeutic conditions. The salt dependence for RNA duplex stability is similar for TAMS, internal loops, and Watson-Crick duplexes. A unified model for predicting the free energy of an RNA duplex with or without loops and mismatches at lower sodium concentrations is presented. The destabilizing effects of PEG 200 are larger for TAMS than internal loops or Watson-Crick duplexes, which may result from different base stacking conformations, dynamics, and water hydration. In contrast, LNA substitutions stabilize internal loops much more than TAMS. Surprisingly, the average per adenosine increase in stability for LNA substitutions in internal loops is -1.82 kcal/mol and only -0.20 kcal/mol for TAMS. The stabilities of TAMS and internal loops with LNA substitutions have similar favorable free energies. Thus, the unfavorable free energy of adenosine internal loops is largely an entropic effect. The favorable stabilities of TAMS result mainly from base stacking. The ability of RNA duplexes to form extended terminal mismatches in the absence of proteins such as argonaute and identifying the enthalpic contributions to terminal mismatch stabilities provide insight into the physical basis of microRNA-mRNA molecular recognition and specificity.

As an instructor, how do you engage the learners? This paper illustrates the value of using learner-centered teaching strategies to accomplish this goal in your classroom. Learner-centered teaching utilizes the instructor as a facilitator and creates an active learning environment in which the student takes responsibility of learning. Various techniques will be described as well as the practical application of leaner-centered teaching in the classroom.

The secondary structure of encapsidated MS2 genomic RNA poses an interesting RNA folding challenge. Cryoelectron microscopy has demonstrated that encapsidated MS2 RNA is well-ordered. Models of MS2 assembly suggest that the RNA hairpin-protein interactions and the appropriate placement of hairpins in the MS2 RNA secondary structure can guide the formation of the correct icosahedral particle. The RNA hairpin motif that is recognized by the MS2 capsid protein dimers, however, is energetically unfavorable, and thus free energy predictions are biased against this motif. Computer programs called Crumple, Sliding Windows, and Assembly provide useful tools for prediction of viral RNA secondary structures when the traditional assumptions of RNA structure prediction by free energy minimization may not apply. These methods allow incorporation of global features of the RNA fold and motifs that are difficult to include directly in minimum free energy predictions. For example, with MS2 RNA the experimental data from SELEX experiments, crystallography, and theoretical calculations of the path for the series of hairpins can be incorporated in the RNA structure prediction, and thus the influence of free energy considerations can be modulated. This approach thoroughly explores conformational space and generates an ensemble of secondary structures. The predictions from this new approach can test hypotheses and models of viral assembly and guide construction of complete three-dimensional models of virus particles.

Resistance mutations to antibiotics targeting rRNA can be far from the drug-binding site. Crystallography studies revealed that the antibiotic resistance mutation G2482A (G2447A in E. coli ) in Haloarcula marismortui 50S ribosomes does not directly contact the drug or introduce changes to the ribosomal structure except for losing a potassium ion coordinated to a base triple at the drug-binding site. Using molecular dynamics simulations, we tested hypotheses regarding the effects of the G2482A mutation and ion coordination on the conformational dynamics of the 50S ribosome. Simulations show that the mutation enhances conformational fluctuation at the antibiotic binding site, weakens the hydrogen-bonding network, and increases flexibility at the 50S peptidyl transferase center (PTC). Our data supports the view that distant mutations can perturb the dynamic network in the ribosomal PTC, thereby raising the entropic cost of antibiotic binding. These results underscore the importance of considering conformational dynamics in rational drug design.

The diverse landscape of RNA conformational space includes many canyons and crevices that are distant from the lowest minimum free energy valley and remain unexplored by traditional RNA structure prediction methods. A complete description of the entire RNA folding landscape can facilitate identification of biologically important conformations. The Crumple algorithm rapidly enumerates all possible non-pseudoknotted structures for an RNA sequence without consideration of thermodynamics while filtering the output with experimental data. The Crumple algorithm provides an alternative approach to traditional free energy minimization programs for RNA secondary structure prediction. A complete computation of all non-pseudoknotted secondary structures can reveal structures that would not be predicted by methods that sample the RNA folding landscape based on thermodynamic predictions. The free energy minimization approach is often successful but is limited by not considering RNA tertiary and protein interactions and the possibility that kinetics rather than thermodynamics determines the functional RNA fold. Efficient parallel computing and filters based on experimental data make practical the complete enumeration of all non-pseudoknotted structures. Efficient parallel computing for Crumple is implemented in a ring graph approach. Filters for experimental data include constraints from chemical probing of solvent accessibility, enzymatic cleavage of paired or unpaired nucleotides, phylogenetic covariation, and the minimum number and lengths of helices determined from crystallography or cryo-electron microscopy. The minimum number and length of helices has a significant effect on reducing conformational space. Pairing constraints reduce conformational space more than single nucleotide constraints. Examples with Alfalfa Mosaic Virus RNA and Trypanosome brucei guide RNA demonstrate the importance of evaluating all possible structures when pseduoknots, RNA-protein interactions, and metastable structures are important for biological function. Crumple software is freely available at http://adenosine.chem.ou.edu/software.html.

Viral genomic RNA adopts many conformations during its life cycle as the genome is replicated, translated, and encapsidated. The high-resolution crystallographic structure of the satellite tobacco mosaic virus (STMV) particle reveals 30 helices of well-ordered RNA. The crystallographic data provide global constraints on the possible secondary structures for the encapsidated RNA. Traditional free energy minimization methods of RNA secondary structure prediction do not generate structures consistent with the crystallographic data, and to date no complete STMV RNA basepaired secondary structure has been generated. RNA-protein interactions and tertiary interactions may contribute a significant degree of stability, and the kinetics of viral assembly may dominate the folding process. The computational tools, Helix Find & Combine, Crumple, and Sliding Windows and Assembly, evaluate and explore the possible secondary structures for encapsidated STMV RNA. All possible hairpins consistent with the experimental data and a cotranscriptional folding and assembly hypothesis were generated, and the combination of hairpins that was most consistent with experimental data is presented as the best representative structure of the ensemble. Multiple solutions to the genome packaging problem could be an evolutionary advantage for viruses. In such cases, an ensemble of structures that share favorable global features best represents the RNA fold., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Prohead RNA (pRNA) is an essential component of the self-assembling ϕ29 bacteriophage DNA packaging motor. Different related species of bacteriophage share only 12% similarity in pRNA sequences. The secondary structure for pRNA is conserved, however. In this study, we present evidence for self-assembly in different pRNA sequences and new measurements of the energetics for the quaternary interactions in pRNA dimers and trimers. The energetics for self-assembly in different pRNA sequences are similar despite very different sequences in the loop-loop interactions. The architecture surrounding the interlocking loops contributes to the stability of the pRNA quaternary interactions, and sequence variation outside the interlocking loops may counterbalance the changes in the loop sequences. Thus, the evolutionary divergence of pRNA sequences maintains not only conservation of function and secondary structure but also stabilities of quaternary interactions. The self-assembly of pRNA can be fine-tuned with variations in magnesium chloride, sodium chloride, temperature, and concentration. The ability to control pRNA self-assembly holds promise for the development of nanoparticle therapeutic applications for this biological molecule. The pRNA system is well suited for future studies to further understand the energetics of RNA tertiary and quaternary interactions, which can provide insight into larger biological assemblies such as viruses and biomolecular motors.

Consecutive GU pairs at the ends of RNA helices provide significant thermodynamic stability between -1.0 and -3.8 kcal/mol at 37 °C, which is equivalent to approximately 2 orders of magnitude in the value of a binding constant. The thermodynamic stabilities of GU pairs depend on the sequence, stacking orientation, and position in the helix. In contrast to GU pairs in the middle of a helix that may be destabilizing, all consecutive terminal GU pairs contribute favorable thermodynamic stability. This work presents measured thermodynamic stabilities for 30 duplexes containing two, three, or four consecutive GU pairs at the ends of RNA helices and a model to predict the thermodynamic stabilities of terminal GU pairs. Imino proton NMR spectra show that the terminal GU nucleotides form hydrogen-bonded pairs. Different orientations of terminal GU pairs can have different conformations with equivalent thermodynamic stabilities. These new data and prediction model will help improve RNA secondary structure prediction, identification of miRNA target sequences with GU pairs, and efforts to understand the fundamental physical forces directing RNA structure and energetics.

The Bacillus subtilis phage phi29 packaging motor requires prohead RNA for genome encapsidation. The nuclear magnetic resonance structure of the prohead RNA E-loop hairpin, r(5'AUUGAGUU), is presented and compared to predictions from MC-SYM. The prohead RNA E-loop hairpins contain sequences similar to rRNA hairpins. Comparison of predicted and experimentally determined prohead and ribosomal hairpin structures reveals that sequence similarity is a stronger determinant of hairpin structural similarity than grouping similar types of RNA. All the hairpins contain a U-turn motif but differ in the first noncanonical pair and backbone orientation. These structures provide benchmarks for further improvements in RNA structure predictions from sequence.

Optical melting experiments provide measurements of thermodynamic parameters for nucleic acids. These thermodynamic parameters are widely used in RNA structure prediction programs and DNA primer design software. This review briefly summarizes the theory and underlying assumptions of the method and provides practical details for instrument calibration, experimental design, and data interpretation., (Copyright © 2009 Elsevier Inc. All rights reserved.)

The thermodynamic stabilities of consecutive mismatches at the ends of RNA helices are determined by the 3' terminal nucleotides. More than 40 RNA duplexes containing terminal motifs of 3 or more nucleotides were studied by optical melting experiments. Up to three noncanonical pairs of nucleotides at the end of RNA helices provide additional thermodynamic stability. 3' nucleotides contribute more stability than 5' nucleotides, and purines contribute more stability than pyrimidines. The additional stability of a second or third 3' nucleotide stacking on a purine is the same for both dangling ends and consecutive terminal mismatches. Current predictions underestimate RNA duplex stabilities with terminal motifs by 1.4 kcal/mol on average, which is an order of magnitude in a binding constant at 37 degrees C. Accurate thermodynamic parameters for these terminal motifs will contribute to improvements in RNA secondary structure predictions, identification of microRNA targets, and design of siRNA therapeutics with fewer off-target effects.

Eleven mutations that make Haloarcula marismortui resistant to anisomycin, an antibiotic that competes with the amino acid side chains of aminoacyl tRNAs for binding to the A-site cleft of the large ribosomal unit, have been identified in 23S rRNA. The correlation observed between the sensitivity of H. marismortui to anisomycin and the affinity of its large ribosomal subunits for the drug indicates that its response to anisomycin is determined primarily by the binding of the drug to its large ribosomal subunit. The structures of large ribosomal subunits containing resistance mutations show that these mutations can be divided into two classes: (1) those that interfere with specific drug-ribosome interactions and (2) those that stabilize the apo conformation of the A-site cleft of the ribosome relative to its drug-bound conformation. The conformational effects of some mutations of the second kind propagate through the ribosome for considerable distances and are reversed when A-site substrates bind to the ribosome.

Negamycin, a small-molecule inhibitor of protein synthesis, binds the Haloarcula marismortui 50S ribosomal subunit at a single site formed by highly conserved RNA nucleotides near the cytosolic end of the nascent chain exit tunnel. The mechanism of antibiotic action and the function of this unexplored tunnel region remain intriguingly elusive.

Crystal structures of the 50 S ribosomal subunit from Haloarcula marismortui complexed with two antibiotics have identified new sites at which antibiotics interact with the ribosome and inhibit protein synthesis. 13-Deoxytedanolide binds to the E site of the 50 S subunit at the same location as the CCA of tRNA, and thus appears to inhibit protein synthesis by competing with deacylated tRNAs for E site binding. Girodazole binds near the E site region, but is somewhat buried and may inhibit tRNA binding by interfering with conformational changes that occur at the E site. The specificity of 13-deoxytedanolide for eukaryotic ribosomes is explained by its extensive interactions with protein L44e, which is an E site component of archaeal and eukaryotic ribosomes, but not of eubacterial ribosomes. In addition, protein L28, which is unique to the eubacterial E site, overlaps the site occupied by 13-deoxytedanolide, precluding its binding to eubacterial ribosomes. Girodazole is specific for eukarytes and archaea because it makes interactions with L15 that are not possible in eubacteria.

The NMR structures of [see text] and [see text] are reported. The internal loop, [see text], is about 2 kcal/mol more stable than [see text] at 37 degrees C. The duplexes assemble into similar global folds characterized by the formation of tandem sheared GA pairs. The different stabilities of the loops are accompanied by differences in the local structure of the closing GU pairs. In the [see text] internal loop, the GU pairs form canonical wobble configurations with two hydrogen bonds, whereas in [see text], the GU pairs form a single hydrogen bond involving the amino group, GH22, and the carbonyl group, UO4. This pairing is similar to the GU closing pair of the 690 hairpin loop found in E. coli 16S rRNA. The [see text] and [see text] structures reveal how the subtle interplay between stacking and hydrogen bonding determines sequence dependent conformation and thermodynamic stability. Thus, this work provides structural and thermodynamic benchmarks for theoreticians in the ongoing effort to understand the sequence dependence of RNA physicochemical properties.

A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%. This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28% to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification contain on average 84% of known canonical base pairs.

The J4/5 loop of the group I intron in the mouse-derived fungal pathogen Pneumocystis carinii is the docking site for the first step of the RNA-catalyzed self-splicing reaction and thus is a model of a potential drug target. This purine-rich asymmetric internal loop, 5'GGAAG/3'UAGU, is also thermodynamically more stable than other internal loops with two GU closing pairs and three nucleotides opposite two nucleotides. The results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments suggest that the GU closing pairs form and that sheared GA pairs form in the internal loop. The NMR spectra show evidence of conformational dynamics, and several GA pairings are possible. Thus, this dynamic loop presents several possible structures for potential binding of drugs that target group I self-splicing introns. The results also contribute to understanding the structural and dynamic basis for the function and thermodynamic stability of this loop.

The 5'(rGGCAAGCCU)(2) duplex contains tandem A.A pairs. The three-dimensional structure of the 5'(rGGCAAGCCU)(2) duplex was modeled by molecular dynamics and energy minimization with NMR-derived distance and dihedral angle restraints. Although the 5'(rCAAG)(2) loop is thermodynamically destabilizing by 1.1 kcal/mol, the tandem A.A pairs adopt a predominant conformation: a sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment similar to that observed in the crystal structure of the P4-P6 domain of the Tetrahymena thermophila intron [Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685]. The NMR-derived structure of the 5'(rGGCAAGCCU)(2) duplex exhibits cross-strand hydrogen bonds from N3 of A4 to an amino hydrogen of A5 and from the 2' oxygen of the A4 sugar to the other amino hydrogen of A5. An intrastrand hydrogen bond is formed from the 2' OH hydrogen of A4 to O5' of A5. The cross-strand A5 bases are stacked. The Watson-Crick G-C regions are essentially A-form. The sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment provides potential contact sites for tertiary interactions and, therefore, is a possible target site for therapeutics. Thus, thermodynamically destabilizing internal loops can be preorganized for tertiary interactions or ligand binding.

Many internal loops that form tertiary contacts in natural RNAs have GU closing pairs; examples include the tetraloop receptor and P1 helix docking site in group I introns. Thus, thermodynamic parameters of internal loops with GU closing pairs can contribute to the prediction of both secondary and tertiary structure. Oligoribonucleotide duplexes containing small internal loops with GU closing pairs were studied by optical melting, one-dimensional imino proton NMR, and one-dimensional phosphorus NMR. The thermodynamic stabilities of asymmetric internal loops with GU closing pairs relative to those of loops with GC closing pairs may be explained by hydrogen bonds. In contrast, the free energy increments for symmetric internal loops of two noncanonical pairs with GU closing pairs relative to loops with GC closing pairs show much more sequence dependence. Imino proton and phosphorus NMR spectra suggest that some GA pairs adjacent to GU closing pairs may form an overall thermodynamically stable but non-A-form conformation.

Optical melting experiments were used to determine the thermodynamic parameters for oligoribonucleotides containing small asymmetric internal loops. The results show a broad range of thermodynamic stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR experiments provide evidence for possible hydrogen bonding in GA and UU mismatches in some asymmetric loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodynamic stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 x 3 and 1 x 3 internal loops.

The energetics of small internal loops are important for prediction of RNA secondary and tertiary structure, selection of drug target sites, and understanding RNA structure-function relationships. Hydrogen bonding, base stacking, electrostatic interactions, backbone distortion, and base-pair size compatibility all contribute to the energetics of small internal loops. Thus, the sequence dependence of these energetics are idiosyncratic. Current approximations for predicting the free energies of internal loops consider size, asymmetry, closing base pairs, and the potential to form GA, GG, or UU pairs. The database of known three-dimensional structures allows for comparison with the models used for predicting stability from sequence., (Copyright 2001 John Wiley & Sons, Inc.)

Improved thermodynamic parameters for prediction of RNA duplex formation are derived from optical melting studies of 90 oligoribonucleotide duplexes containing only Watson-Crick base pairs. To test end or base composition effects, new sets of duplexes are included that have identical nearest neighbors, but different base compositions and therefore different ends. Duplexes with terminal GC pairs are more stable than duplexes with the same nearest neighbors but terminal AU pairs. Penalizing terminal AU base pairs by 0.45 kcal/mol relative to terminal GC base pairs significantly improves predictions of DeltaG degrees37 from a nearest-neighbor model. A physical model is suggested in which the differential treatment of AU and GC ends accounts for the dependence of the total number of Watson-Crick hydrogen bonds on the base composition of a duplex. On average, the new parameters predict DeltaG degrees37, DeltaH degrees, DeltaS degrees, and TM within 3.2%, 6.0%, 6.8%, and 1.3 degreesC, respectively. These predictions are within the limit of the model, based on experimental results for duplexes predicted to have identical thermodynamic parameters.

The vestibular, proprioceptive and visual senses of the body are all affected by alterations of the normal sensory environment during weightlessness. This study was designed to observe muscle activation characteristics when a single component of the sensory environment was altered. Partially immersing a subject in a water pool provided a buoyant force upon the lower body, "unloading" the muscles, similar to the effect on the muscles in weightlessness. Surface EMG from the soleus was obtained during the performance of a constant-force isometric contraction. The mean and median characteristic frequencies were calculated from the power spectrum of each trial. Six of ten subjects showed a difference in the characteristic frequencies between the two environments. It appears that for some individuals there are changes in muscle activation characteristics due to influences of the proprioceptive system when exposed to an altered sensory environment.