High-Throughput Investigation of Diverse Junction Elements in RNA Tertiary Folding.

RNAs fold into defined tertiary structures to function in critical biological processes. While quantitative models can predict RNA secondary structure stability, we are still unable to predict the thermodynamic stability of RNA tertiary structure. Here, we probe conformational preferences of diverse RNA two-way junctions to develop a predictive model for the formation of RNA tertiary structure. We quantitatively measured tertiary assembly energetics of >1,000 of RNA junctions inserted in multiple structural scaffolds to generate a "thermodynamic fingerprint" for each junction. Thermodynamic fingerprints enabled comparison of junction conformational preferences, revealing principles for how sequence influences 3-dimensional conformations. Utilizing fingerprints of junctions with known crystal structures, we generated ensembles for related junctions that predicted their thermodynamic effects on assembly formation. This work reveals sequence-structure-energetic relationships in RNA, demonstrates the capacity for diverse compensation strategies within tertiary structures, and provides a path to quantitative modeling of RNA folding energetics based on "ensemble modularity."

The roles of structural dynamics in the cellular functions of RNAs.

RNAs fold into 3D structures that range from simple helical elements to complex tertiary structures and quaternary ribonucleoprotein assemblies. The functions of many regulatory RNAs depend on how their 3D structure changes in response to a diverse array of cellular conditions. In this Review, we examine how the structural characterization of RNA as dynamic ensembles of conformations, which form with different probabilities and at different timescales, is improving our understanding of RNA function in cells. We discuss the mechanisms of gene regulation by microRNAs, riboswitches, ribozymes, post-transcriptional RNA modifications and RNA-binding proteins, and how the cellular environment and processes such as liquid-liquid phase separation may affect RNA folding and activity. The emerging RNA-ensemble-function paradigm is changing our perspective and understanding of RNA regulation, from in vitro to in vivo and from descriptive to predictive.

Understanding nucleic acid-ion interactions.

Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be understood without a reasonably sophisticated appreciation of these ions' electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid-ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid-ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid-ion interactions.

RNA conformational propensities determine cellular activity.

Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes1. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend both on the strength of the contacts and on the inherent propensities to form binding-competent conformational states2,3. Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions4,5. However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities affect cellular activity. Here we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state.

A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins.

High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.

Biological phosphoryl-transfer reactions: understanding mechanism and catalysis.

Phosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field.

Dissecting the energetic architecture within an RNA tertiary structural motif via high-throughput thermodynamic measurements.

Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact "motifs," whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here, we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base-paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability-abundance relationship for 11ntR sequence variants. These findings of "exceptions to the rule" highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.

Decoupling of catalysis and transition state analog binding from mutations throughout a phosphatase revealed by high-throughput enzymology.

Using high-throughput microfluidic enzyme kinetics (HT-MEK), we measured over 9,000 inhibition curves detailing impacts of 1,004 single-site mutations throughout the alkaline phosphatase PafA on binding affinity for two transition state analogs (TSAs), vanadate and tungstate. As predicted by catalytic models invoking transition state complementary, mutations to active site and active-site-contacting residues had highly similar impacts on catalysis and TSA binding. Unexpectedly, most mutations to more distal residues that reduced catalysis had little or no impact on TSA binding and many even increased tungstate affinity. These disparate effects can be accounted for by a model in which distal mutations alter the enzyme's conformational landscape, increasing the occupancy of microstates that are catalytically less effective but better able to accommodate larger transition state analogs. In support of this ensemble model, glycine substitutions (rather than valine) were more likely to increase tungstate affinity (but not more likely to impact catalysis), presumably due to increased conformational flexibility that allows previously disfavored microstates to increase in occupancy. These results indicate that residues throughout an enzyme provide specificity for the transition state and discriminate against analogs that are larger only by tenths of an Ångström. Thus, engineering enzymes that rival the most powerful natural enzymes will likely require consideration of distal residues that shape the enzyme's conformational landscape and fine-tune active-site residues. Biologically, the evolution of extensive communication between the active site and remote residues to aid catalysis may have provided the foundation for allostery to make it a highly evolvable trait.

High-throughput dissection of the thermodynamic and conformational properties of a ubiquitous class of RNA tertiary contact motifs.

Despite RNA's diverse secondary and tertiary structures and its complex conformational changes, nature utilizes a limited set of structural "motifs"-helices, junctions, and tertiary contact modules-to build diverse functional RNAs. Thus, in-depth descriptions of a relatively small universe of RNA motifs may lead to predictive models of RNA tertiary conformational landscapes. Motifs may have different properties depending on sequence and secondary structure, giving rise to subclasses that expand the universe of RNA building blocks. Yet we know very little about motif subclasses, given the challenges in mapping conformational properties in high throughput. Previously, we used "RNA on a massively parallel array" (RNA-MaP), a quantitative, high-throughput technique, to study thousands of helices and two-way junctions. Here, we adapt RNA-MaP to study the thermodynamic and conformational properties of tetraloop/tetraloop receptor (TL/TLR) tertiary contact motifs, analyzing 1,493 TLR sequences from different classes. Clustering analyses revealed variability in TL specificity, stability, and conformational behavior. Nevertheless, natural GAAA/11ntR TL/TLRs, while varying in tertiary stability by ∼2.5 kcal/mol, exhibited conserved TL specificity and conformational properties. Thus, RNAs may tune stability without altering the overall structure of these TL/TLRs. Furthermore, their stability correlated with natural frequency, suggesting thermodynamics as the dominant selection pressure. In contrast, other TL/TLRs displayed heterogenous conformational behavior and appear to not be under strong thermodynamic selection. Our results build toward a generalizable model of RNA-folding thermodynamics based on the properties of isolated motifs, and our characterized TL/TLR library can be used to engineer RNAs with predictable thermodynamic and conformational behavior.

Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles.

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme's reaction cycle. To examine positioning and motions and test catalytic proposals, we collected "room temperature" X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI's reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI's general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure-function relationships, providing the basis for a new era of "ensemble-function" interrogation of enzymes.

Direct Measurement of Interhelical DNA Repulsion and Attraction by Quantitative Cross-Linking.

To better understand the forces that mediate nucleic acid compaction in biology, we developed the disulfide cross-linking approach xHEED (X-linking of Helices to measure Electrostatic Effects at Distance) to measure the distance-dependent encounter frequency of two DNA helices in solution. Using xHEED, we determined the distance that the electrostatic potential extends from DNA helices, the dependence of this distance on ionic conditions, and the magnitude of repulsion when two helices approach one another. Across all conditions tested, the potential falls to that of the bulk solution within 15 Å of the major groove surface. For separations of ∼30 Å, we measured a repulsion of 1.8 kcal/mol in low monovalent ion concentration (30 mM Na+), with higher Na+ concentrations ameliorating this repulsion, and 2 M Na+ or 100 mM Mg2+ eliminating it. Strikingly, we found full screening at very low Co3+ concentrations and net attraction at higher concentrations, without the higher-order DNA condensation that typically complicates studies of helical attraction. Our measurements define the relevant distances for electrostatic interactions of nucleic-acid helices in biology and introduce a new method to propel further understanding of how these forces impact biological processes.

The structural ensemble of a Holliday junction determined by X-ray scattering interference.

The DNA four-way (Holliday) junction is the central intermediate of genetic recombination, yet key aspects of its conformational and thermodynamic properties remain unclear. While multiple experimental approaches have been used to characterize the canonical X-shape conformers under specific ionic conditions, the complete conformational ensemble of this motif, especially at low ionic conditions, remains largely undetermined. In line with previous studies, our single-molecule Förster resonance energy transfer (smFRET) measurements of junction dynamics revealed transitions between two states under high salt conditions, but smFRET could not determine whether there are fast and unresolvable transitions between distinct conformations or a broad ensemble of related states under low and intermediate salt conditions. We therefore used an emerging technique, X-ray scattering interferometry (XSI), to directly probe the conformational ensemble of the Holliday junction across a wide range of ionic conditions. Our results demonstrated that the four-way junction adopts an out-of-plane geometry under low ionic conditions and revealed a conformational state at intermediate ionic conditions previously undetected by other methods. Our results provide critical information to build toward a full description of the conformational landscape of the Holliday junction and underscore the utility of XSI for probing conformational ensembles under a wide range of solution conditions.

Blind tests of RNA-protein binding affinity prediction.

Interactions between RNA and proteins are pervasive in biology, driving fundamental processes such as protein translation and participating in the regulation of gene expression. Modeling the energies of RNA-protein interactions is therefore critical for understanding and repurposing living systems but has been hindered by complexities unique to RNA-protein binding. Here, we bring together several advances to complete a calculation framework for RNA-protein binding affinities, including a unified free energy function for bound complexes, automated Rosetta modeling of mutations, and use of secondary structure-based energetic calculations to model unbound RNA states. The resulting Rosetta-Vienna RNP-ΔΔG method achieves root-mean-squared errors (RMSEs) of 1.3 kcal/mol on high-throughput MS2 coat protein-RNA measurements and 1.5 kcal/mol on an independent test set involving the signal recognition particle, human U1A, PUM1, and FOX-1. As a stringent test, the method achieves RMSE accuracy of 1.4 kcal/mol in blind predictions of hundreds of human PUM2-RNA relative binding affinities. Overall, these RMSE accuracies are significantly better than those attained by prior structure-based approaches applied to the same systems. Importantly, Rosetta-Vienna RNP-ΔΔG establishes a framework for further improvements in modeling RNA-protein binding that can be tested by prospective high-throughput measurements on new systems.

Sequence-dependent RNA helix conformational preferences predictably impact tertiary structure formation.

Structured RNAs and RNA complexes underlie biological processes ranging from control of gene expression to protein translation. Approximately 50% of nucleotides within known structured RNAs are folded into Watson-Crick (WC) base pairs, and sequence changes that preserve these pairs are typically assumed to preserve higher-order RNA structure and binding of macromolecule partners. Here, we report that indirect effects of the helix sequence on RNA tertiary stability are, in fact, significant but are nevertheless predictable from a simple computational model called RNAMake-∆∆G. When tested through the RNA on a massively parallel array (RNA-MaP) experimental platform, blind predictions for >1500 variants of the tectoRNA heterodimer model system achieve high accuracy (rmsd 0.34 and 0.77 kcal/mol for sequence and length changes, respectively). Detailed comparison of predictions to experiments support a microscopic picture of how helix sequence changes subtly modulate conformational fluctuations at each base-pair step, which accumulate to impact RNA tertiary structure stability. Our study reveals a previously overlooked phenomenon in RNA structure formation and provides a framework of computation and experiment for understanding helix conformational preferences and their impact across biological RNA and RNA-protein assemblies.

Demonstration of protein cooperativity mediated by RNA structure using the human protein PUM2.

Posttranslational gene regulation requires a complex network of RNA-protein interactions. Cooperativity, which tunes response sensitivities, originates from protein-protein interactions in many systems. For RNA-binding proteins, cooperativity can also be mediated through RNA structure. RNA structural cooperativity (RSC) arises when binding of one protein induces a redistribution of RNA conformational states that enhance access (positive cooperativity) or block access (negative cooperativity) to additional binding sites. As RSC does not require direct protein-protein interactions, it allows cooperativity to be tuned for individual RNAs, via alterations in sequence that alter structural stability. Given the potential importance of this mechanism of control and our desire to quantitatively dissect features that underlie physiological regulation, we developed a statistical mechanical framework for RSC and tested this model by performing equilibrium binding measurements of the human PUF family protein PUM2. Using 68 RNAs that contain two to five PUM2-binding sites and RNA structures of varying stabilities, we observed a range of structure-dependent cooperative behaviors. To test our ability to account for this cooperativity with known physical constants, we used PUM2 affinity and nearest-neighbor RNA secondary structure predictions. Our model gave qualitative agreement for our disparate set of 68 RNAs across two temperatures, but quantitative deviations arise from overestimation of RNA structural stability. Our results demonstrate cooperativity mediated by RNA structure and underscore the power of quantitative stepwise experimental evaluation of mechanisms and computational tools.

Cation enrichment in the ion atmosphere is promoted by local hydration of DNA.

Electrostatic interactions are central to the structure and function of nucleic acids, including their folding, condensation, and interaction with proteins and other charged molecules. These interactions are profoundly affected by ions surrounding nucleic acids, the constituents of the so-called ion atmosphere. Here, we report precise Fourier Transform-Terahertz/Far-Infrared (FT-THz/FIR) measurements in the frequency range 30-500 cm-1 for a 24-bp DNA solvated in a series of alkali halide (NaCl, NaF, KCl, CsCl, and CsF) electrolyte solutions which are sensitive to changes in the ion atmosphere. Cation excess in the ion atmosphere is detected experimentally by observation of cation modes of Na+, K+, and Cs+ in the frequency range between 70-90 cm-1. Based on MD simulations, we propose that the magnitude of cation excess (which is salt specific) depends on the ability of the electrolyte to perturb the water network at the DNA interface: In the NaF atmosphere, the ions reduce the strength of interactions between water and the DNA more than in case of a NaCl electrolyte. Here, we explicitly take into account the solvent contribution to the chemical potential in the ion atmosphere: A decrease in the number of bound water molecules in the hydration layer of DNA is correlated with enhanced density fluctuations, which decrease the free energy cost of ion-hydration, thus promoting further ion accumulation within the DNA atmosphere. We propose that taking into account the local solvation is crucial for understanding the ion atmosphere.

Increasing the length of poly-pyrimidine bulges broadens RNA conformational ensembles with minimal impact on stacking energetics.

Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of noncoding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1-4, 7) across a range of Mg2+ concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg2+, helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population <15%), whereas one-bulge and two-bulge motifs adopt predominantly stacked conformations (stacked population >74%). In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population >84%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition is within threefold regardless of bulge length. In the absence of Mg2+, the difference between free energy of interhelical coaxial stacking across the bulge variants is estimated to be ∼2.9 kcal/mol, based on an NMR chemical shift mapping with stacking being more energetically disfavored for the longer bulges. This difference decreases to ∼0.4 kcal/mol in the presence of Mg2+ NMR RDCs and resonance intensity data show increased dynamics in the stacked state with increasing bulge length in the presence of Mg2+ We propose that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thereby increasing the number of coaxial conformations that are sampled. Energetically compensated interhelical stacking dynamics may help to maximize the conformational adaptability of RNA and allow a wide range of conformations to be optimally stabilized by proteins and ligands.

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics.

Decades of study of the architecture and function of structured RNAs have led to the perspective that RNA tertiary structure is modular, made of locally stable domains that retain their structure across RNAs. We formalize a hypothesis inspired by this modularity-that RNA folding thermodynamics and kinetics can be quantitatively predicted from separable energetic contributions of the individual components of a complex RNA. This reconstitution hypothesis considers RNA tertiary folding in terms of ΔGalign, the probability of aligning tertiary contact partners, and ΔGtert, the favorable energetic contribution from the formation of tertiary contacts in an aligned state. This hypothesis predicts that changes in the alignment of tertiary contacts from different connecting helices and junctions (ΔGHJH) or from changes in the electrostatic environment (ΔG+/-) will not affect the energetic perturbation from a mutation in a tertiary contact (ΔΔGtert). Consistent with these predictions, single-molecule FRET measurements of folding of model RNAs revealed constant ΔΔGtert values for mutations in a tertiary contact embedded in different structural contexts and under different electrostatic conditions. The kinetic effects of these mutations provide further support for modular behavior of RNA elements and suggest that tertiary mutations may be used to identify rate-limiting steps and dissect folding and assembly pathways for complex RNAs. Overall, our model and results are foundational for a predictive understanding of RNA folding that will allow manipulation of RNA folding thermodynamics and kinetics. Conversely, the approaches herein can identify cases where an independent, additive model cannot be applied and so require additional investigation.

Quantitative Studies of an RNA Duplex Electrostatics by Ion Counting.

RNAs are one of the most charged polyelectrolytes in nature, and understanding their electrostatics is fundamental to their structure and biological functions. An effective way to characterize the electrostatic field generated by nucleic acids is to quantify interactions between nucleic acids and ions that surround the molecules. These ions form a loosely associated cloud referred to as an ion atmosphere. Although theoretical and computational studies can describe the ion atmosphere around RNAs, benchmarks are needed to guide the development of these approaches, and experiments to date that read out RNA-ion interactions are limited. Here, we present ion counting studies to quantify the number of ions surrounding well-defined model systems of RNA and DNA duplexes. We observe that the RNA duplex attracts more cations and expels fewer anions compared to the DNA duplex, and the RNA duplex interacts significantly stronger with the divalent cation Mg2+, despite their identical total charge. These experimental results suggest that the RNA duplex generates a stronger electrostatic field than DNA, as is predicted based on the structural differences between their helices. Theoretical calculations using a nonlinear Poisson-Boltzmann equation give excellent agreement with experiments for monovalent ions but underestimate Mg2+-DNA and Mg2+-RNA interactions by 20%. These studies provide needed stringent benchmarks to use against other all-atom theoretical models of RNA-ion interactions, interactions that likely must be accurately accounted for in structural, dynamic, and energetic terms to confidently model RNA structure, interactions, and function.