Atomistic structure of the SARS-CoV-2 pseudoknot in solution from SAXS-driven molecular dynamics.

SARS-CoV-2 depends on -1 programmed ribosomal frameshifting (-1 PRF) to express proteins essential for its replication. The RNA pseudoknot stimulating -1 PRF is thus an attractive drug target. However, the structural models of this pseudoknot obtained from cryo-EM and crystallography differ in some important features, leaving the pseudoknot structure unclear. We measured the solution structure of the pseudoknot using small-angle X-ray scattering (SAXS). The measured profile did not agree with profiles computed from the previously solved structures. Beginning with each of these solved structures, we used the SAXS data to direct all atom molecular dynamics (MD) simulations to improve the agreement in profiles. In all cases, this refinement resulted in a bent conformation that more closely resembled the cryo-EM structures than the crystal structure. Applying the same approach to a point mutant abolishing -1 PRF revealed a notably more bent structure with reoriented helices. This work clarifies the dynamic structures of the SARS-CoV-2 pseudoknot in solution.

Structural analyses of an RNA stability element interacting with poly(A).

Cis-acting RNA elements are crucial for the regulation of polyadenylated RNA stability. The element for nuclear expression (ENE) contains a U-rich internal loop flanked by short helices. An ENE stabilizes RNA by sequestering the poly(A) tail via formation of a triplex structure that inhibits a rapid deadenylation-dependent decay pathway. Structure-based bioinformatic studies identified numerous ENE-like elements in evolutionarily diverse genomes, including a subclass containing two ENE motifs separated by a short double-helical region (double ENEs [dENEs]). Here, the structure of a dENE derived from a rice transposable element (TWIFB1) before and after poly(A) binding (∼24 kDa and ∼33 kDa, respectively) is investigated. We combine biochemical structure probing, small angle X-ray scattering (SAXS), and cryo-electron microscopy (cryo-EM) to investigate the dENE structure and its local and global structural changes upon poly(A) binding. Our data reveal 1) the directionality of poly(A) binding to the dENE, and 2) that the dENE-poly(A) interaction involves a motif that protects the 3'-most seven adenylates of the poly(A). Furthermore, we demonstrate that the dENE does not undergo a dramatic global conformational change upon poly(A) binding. These findings are consistent with the recently solved crystal structure of a dENE+poly(A) complex [S.-F. Torabi et al., Science 371, eabe6523 (2021)]. Identification of additional modes of poly(A)-RNA interaction opens new venues for better understanding of poly(A) tail biology.

Solution structure(s) of trinucleosomes from contrast variation SAXS.

Nucleosomes in all eukaryotic cells are organized into higher order structures that facilitate genome compaction. Visualizing these organized structures is an important step in understanding how genomic DNA is efficiently stored yet remains accessible to information-processing machinery. Arrays of linked nucleosomes serve as useful models for understanding how the properties of both DNA and protein partners affect their arrangement. A number of important questions are also associated with understanding how the spacings between nucleosomes are affected by the histone proteins, chromatin remodelers, or other chromatin-associated protein partners. Contrast variation small angle X-ray scattering (CVSAXS) reports the DNA conformation within protein-DNA complexes and here is applied to measure the conformation(s) of trinucleosomes in solution, with specific sensitivity to the distance between and relative orientation of linked nucleosomes. These data are interpreted in conjunction with DNA models that account for its sequence dependent mechanical properties, and Monte-Carlo techniques that generate realistic structures for comparison with measured scattering profiles. In solution, trinucleosomes segregate into two dominant populations, with the flanking nucleosomes stacked or nearly equilaterally separated, e.g. with roughly equal distance between all pairs of nucleosomes. These populations are consistent with previously observed magnesium-dependent structures of trinucleosomes with shorter linkers.

Visualizing a viral genome with contrast variation small angle X-ray scattering.

Despite the threat to human health posed by some single-stranded RNA viruses, little is understood about their assembly. The goal of this work is to introduce a new tool for watching an RNA genome direct its own packaging and encapsidation by proteins. Contrast variation small-angle X-ray scattering (CV-SAXS) is a powerful tool with the potential to monitor the changing structure of a viral RNA through this assembly process. The proteins, though present, do not contribute to the measured signal. As a first step in assessing the feasibility of viral genome studies, the structure of encapsidated MS2 RNA was exclusively detected with CV-SAXS and compared with a structure derived from asymmetric cryo-EM reconstructions. Additional comparisons with free RNA highlight the significant structural rearrangements induced by capsid proteins and invite the application of time-resolved CV-SAXS to reveal interactions that result in efficient viral assembly.

Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2).

Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 ribosomal frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

Elucidating the Role of Microprocessor Protein DGCR8 in Bending RNA Structures.

Although conformational dynamics of RNA molecules are potentially important in microRNA (miRNA) processing, the role of the protein binding partners in facilitating the requisite structural changes is not well understood. In previous work, we and others have demonstrated that nonduplex structural elements and the conformational flexibility they support are necessary for efficient RNA binding and cleavage by the proteins associated with the two major stages of miRNA processing. However, recent studies showed that the protein DGCR8 binds primary miRNA and duplex RNA with similar affinities. Here, we study RNA binding by a small recombinant construct of the DGCR8 protein and the RNA conformation changes that result. This construct, the DGCR8 core, contains two double-stranded RNA-binding domains (dsRBDs) and a C-terminal tail. To assess conformational changes resulting from binding, we applied small-angle x-ray scattering with contrast variation to detect conformational changes of primary-miR-16-1 in complex with the DGCR8 core. This method reports only on the RNA conformation within the complex and suggests that the protein bends the RNA upon binding. Supporting work using smFRET to study the conformation of RNA duplexes bound to the core also shows bending. Together, these studies elucidate the role of DGCR8 in interacting with RNA during the early stages of miRNA processing.

Visualizing Disordered Single-Stranded RNA: Connecting Sequence, Structure, and Electrostatics.

Disordered homopolymeric regions of single-stranded RNA, such as U or A tracts, are found within functional RNAs where they play distinct roles in defining molecular structure and facilitating recognition by partners. Despite this prominence, details of conformational and biophysical properties of these regions have not yet been resolved. We apply a number of experimental techniques to investigate the conformations of these biologically important motifs and provide quantitative measurements of their ion atmospheres. Single strands of RNA display pronounced sequence-dependent conformations that relate to the unique ion atmospheres each attracts. Chains of rU bases are relatively unstructured under all conditions, while chains of rA bases display distinct ordering through stacking or clustering motifs, depending on the composition of the surrounding solution. These dramatic structural differences are consistent with the measured disparity in ion composition and atmospheres around each homopolymer, revealing a complex interplay of base, ion, and single-strand ordering. The unique structural and ionic signatures of homopolymer ssRNAs explains their role(s) in folding structured RNAs and may explain their distinct recognition by protein partners.

Characterizing Enzyme Reactions in Microcrystals for Effective Mix-and-Inject Experiments using X-ray Free-Electron Lasers.

Mix-and-inject serial crystallography is an emerging technique that utilizes X-ray free-electron lasers (XFELs) and microcrystalline samples to capture atomically detailed snapshots of biomolecules as they function. Early experiments have yielded exciting results; however, there are limited options to characterize reactions in crystallo in advance of the beamtime. Complementary measurements are needed to identify the best conditions and timescales for observing structural intermediates. Here, we describe the interface of XFEL compatible mixing injectors with rapid freeze-quenching and X-band EPR spectroscopy, permitting characterization of reactions in crystals under the same conditions as an XFEL experiment. We demonstrate this technology by tracking the reaction of azide with microcrystalline myoglobin, using only a fraction of the sample required for a mix-and-inject experiment. This spectroscopic method enables optimization of sample and mixer conditions to maximize the populations of intermediate states, eliminating the guesswork of current mix-and-inject experiments.

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure.

Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide Ecoli rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg2+, Ca2+, or Sr2+ together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg2+ to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure. Subsequently, specific divalent ions modulate populations of intermediates in conformational ensembles along the folding pathway with transition times longer than 10 msec. Rate constants for the five folding transitions act on timescales from submillisecond to tens of seconds. The sensitivity of RNA tertiary structure to divalent cation identity affects all but the fastest events in RNA folding, and allowed us to identify those states that prefer Mg2+ The GTPase center RNA appears to have optimized its folding trajectory to specifically utilize this most abundant intracellular divalent ion.

Revealing the distinct folding phases of an RNA three-helix junction.

Remarkable new insight has emerged into the biological role of RNA in cells. RNA folding and dynamics enable many of these newly discovered functions, calling for an understanding of RNA self-assembly and conformational dynamics. Because RNAs pass through multiple structures as they fold, an ensemble perspective is required to visualize the flow through fleetingly populated sets of states. Here, we combine microfluidic mixing technology and small angle X-ray scattering (SAXS) to measure the Mg-induced folding of a small RNA domain, the tP5abc three helix junction. Our measurements are interpreted using ensemble optimization to select atomically detailed structures that recapitulate each experimental curve. Structural ensembles, derived at key stages in both time-resolved studies and equilibrium titrations, reproduce the features of known intermediates, and more importantly, offer a powerful new structural perspective on the time-progression of folding. Distinct collapse phases along the pathway appear to be orchestrated by specific interactions with Mg ions. These key interactions subsequently direct motions of the backbone that position the partners of tertiary contacts for later bonding, and demonstrate a remarkable synergy between Mg and RNA across numerous time-scales.

The ATPase motor of the Chd1 chromatin remodeler stimulates DNA unwrapping from the nucleosome.

Chromatin remodelers are ATP-dependent motors that reorganize DNA packaging by disrupting canonical histone-DNA contacts within the nucleosome. Here, we show that the Chd1 chromatin remodeler stimulates DNA unwrapping from the edge of the nucleosome in a nucleotide-dependent and DNA sequence-sensitive fashion. Nucleosome binding, monitored by stopped flow, was complex and sensitive to nucleotide, with AMP-PNP promoting faster binding than ADP·BeF3-. Nucleosome unwrapping by Chd1, examined by bulk FRET, occurred in the presence and absence of nucleotide and did not require the Chd1 DNA-binding domain. In AMP-PNP conditions, Chd1 unwrapped one side of the Widom 601 DNA more easily than the other, consistent with previous observations of 601 asymmetry and indicating that Chd1 amplifies intrinsic sequence properties of nucleosomal DNA. Using small angle X-ray scattering (SAXS) with contrast variation, we found distinct DNA conformations depending on the nucleotide analog bound to Chd1: with AMP-PNP, DNA primarily unwrapped in-plane with the nucleosomal disk, whereas with ADP·BeF3-, a significant fraction showed distinctive out-of-plane unwrapping as well. Taken together, our findings show tight coupling between entry/exit DNA of the nucleosome and the Chd1 ATPase motor, suggesting that dynamic nucleosome unwrapping is coupled to nucleosome binding and remodeling by Chd1.

Structural changes of tailless bacteriophage ΦX174 during penetration of bacterial cell walls.

Unlike tailed bacteriophages, which use a preformed tail for transporting their genomes into a host bacterium, the ssDNA bacteriophage ΦX174 is tailless. Using cryo-electron microscopy and time-resolved small-angle X-ray scattering, we show that lipopolysaccharides (LPS) form bilayers that interact with ΦX174 at an icosahedral fivefold vertex and induce single-stranded (ss) DNA genome ejection. The structures of ΦX174 complexed with LPS have been determined for the pre- and post-ssDNA ejection states. The ejection is initiated by the loss of the G protein spike that encounters the LPS, followed by conformational changes of two polypeptide loops on the major capsid F proteins. One of these loops mediates viral attachment, and the other participates in making the fivefold channel at the vertex contacting the LPS.

Asymmetric unwrapping of nucleosomal DNA propagates asymmetric opening and dissociation of the histone core.

The nucleosome core particle (NCP) is the basic structural unit for genome packaging in eukaryotic cells and consists of DNA wound around a core of eight histone proteins. DNA access is modulated through dynamic processes of NCP disassembly. Partly disassembled structures, such as the hexasome (containing six histones) and the tetrasome (four histones), are important for transcription regulation in vivo. However, the pathways for their formation have been difficult to characterize. We combine time-resolved (TR) small-angle X-ray scattering and TR-FRET to correlate changes in the DNA conformations with composition of the histone core during salt-induced disassembly of canonical NCPs. We find that H2A-H2B histone dimers are released sequentially, with the first dimer being released after the DNA has formed an asymmetrically unwrapped, teardrop-shape DNA structure. This finding suggests that the octasome-to-hexasome transition is guided by the asymmetric unwrapping of the DNA. The link between DNA structure and histone composition suggests a potential mechanism for the action of proteins that alter nucleosome configurations such as histone chaperones and chromatin remodeling complexes.

Conformations of an RNA Helix-Junction-Helix Construct Revealed by SAXS Refinement of MD Simulations.

RNA is involved in a broad range of biological processes that extend far beyond translation. Many of RNA's recently discovered functions rely on folding to a specific conformation or transitioning between conformations. The RNA structure contains rigid, short basepaired regions connected by more flexible linkers. Studies of model constructs such as small helix-junction-helix (HJH) motifs are useful in understanding how these elements work together to determine RNA conformation. Here, we reveal the full ensemble of solution structures assumed by a model RNA HJH. We apply small-angle x-ray scattering and an ensemble optimization method to selectively refine models generated by all-atom molecular dynamics simulations. The expectation of a broad distribution of helix orientations, at and above physiological ionic strength, is not met. Instead, this analysis shows that the HJH structures are dominated by two distinct conformations at moderate to high ionic strength. Atomic structures, selected from the molecular dynamics simulations, reveal strong base-base interactions in the junction that critically constrain the conformational space available to the HJH molecule and lead to a surprising re-extension at high salt. These results are corroborated by comparison with previous single-molecule fluorescence resonance energy transfer experiments on the same constructs.

Microfluidic Mixing Injector Holder Enables Routine Structural Enzymology Measurements with Mix-and-Inject Serial Crystallography Using X-ray Free Electron Lasers.

The emerging technique of Mix-and-Inject Serial Crystallography (MISC) at X-ray free electron laser sources provides atomically detailed structural information about biomolecules as they function. Despite early successes, MISC is currently limited by the efficiency and robustness of the mixing injectors used to initiate the reaction and propel the sample into the X-ray beam for measurement. Here, we present a new method for fabricating the injector system that leads to simpler, faster, and more effective experiments. A mixing injector can now be produced from raw components in 100 min, only 5 min of which must be spent during the experiment, saving valuable time. The system is modular, enabling parts to be quickly exchanged in the event of unanticipated experimental difficulties, such as clogging. The injector holder is designed to be flexible, allowing each device to be optimized to maximize the number of diffraction patterns measured during each experiment. This holder has been used successfully during four beamtimes at two different X-ray free electron laser sources. Its robustness and ease of use is an important step toward making the MISC technique accessible and routine.

Visualizing single-stranded nucleic acids in solution.

Single-stranded nucleic acids (ssNAs) are ubiquitous in many key cellular functions. Their flexibility limits both the number of high-resolution structures available, leaving only a small number of protein-ssNA crystal structures, while forcing solution investigations to report ensemble averages. A description of the conformational distributions of ssNAs is essential to more fully characterize biologically relevant interactions. We combine small angle X-ray scattering (SAXS) with ensemble-optimization methods (EOM) to dynamically build and refine sets of ssNA structures. By constructing candidate chains in representative dinucleotide steps and refining the models against SAXS data, a broad array of structures can be obtained to match varying solution conditions and strand sequences. In addition to the distribution of large scale structural parameters, this approach reveals, for the first time, intricate details of the phosphate backbone and underlying strand conformations. Such information on unperturbed strands will critically inform a detailed understanding of an array of problems including protein-ssNA binding, RNA folding and the polymer nature of NAs. In addition, this scheme, which couples EOM selection with an iteratively refining pool to give confidence in the underlying structures, is likely extendable to the study of other flexible systems.

The impact of base stacking on the conformations and electrostatics of single-stranded DNA.

Single-stranded DNA (ssDNA) is notable for its interactions with ssDNA binding proteins (SSBs) during fundamentally important biological processes including DNA repair and replication. Previous work has begun to characterize the conformational and electrostatic properties of ssDNA in association with SSBs. However, the conformational distributions of free ssDNA have been difficult to determine. To capture the vast array of ssDNA conformations in solution, we pair small angle X-ray scattering with novel ensemble fitting methods, obtaining key parameters such as the size, shape and stacking character of strands with different sequences. Complementary ion counting measurements using inductively coupled plasma atomic emission spectroscopy are employed to determine the composition of the ion atmosphere at physiological ionic strength. Applying this combined approach to poly dA and poly dT, we find that the global properties of these sequences are very similar, despite having vastly different propensities for single-stranded helical stacking. These results suggest that a relatively simple mechanism for the binding of ssDNA to non-specific SSBs may be at play, which explains the disparity in binding affinities observed for these systems.

Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography.

BACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis ß-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

Local DNA Sequence Controls Asymmetry of DNA Unwrapping from Nucleosome Core Particles.

DNA is tightly wrapped around histone proteins in nucleosome core particles (NCPs) yet must become accessible for processing in the cell. This accessibility, a key component of transcription regulation, is influenced by the properties of both the histone proteins and the DNA itself. Small angle x-ray scattering with contrast variation is used to examine how sequence variations affect DNA unwrapping from NCPs at different salt concentrations. Salt destabilizes NCPs, populating multiple unwrapped states as many possible unwrapping pathways are explored by the complexes. We apply coarse-grained Monte Carlo methods to generate realistic sequence-dependent unwrapped structures for the nucleosomal DNA with thermal variations. An ensemble optimization method is employed to determine the composition of the overall ensemble as electrostatic interactions are weakened. Interesting DNA-sequence-dependent differences are revealed in the unwrapping paths and equilibrium constants. These differences are correlated with specific features within the nucleic acid sequences.

Spermine Condenses DNA, but Not RNA Duplexes.

Interactions between the polyamine spermine and nucleic acids drive important cellular processes. Spermine condenses DNA and some RNAs, such as poly(rA):poly(rU). A large fraction of the spermine present in cells is bound to RNA but apparently does not condense it. Here, we study the effect of spermine binding to short duplex RNA and DNA, and compare our findings with predictions of molecular-dynamics simulations. When small numbers of spermine are introduced, RNA with a designed sequence containing a mixture of 14 GC pairs and 11 AU pairs resists condensation relative to DNA of an equivalent sequence or to 25 bp poly(rA):poly(rU) RNA. A comparison of wide-angle x-ray scattering profiles with simulation results suggests that spermine is sequestered deep within the major groove of mixed-sequence RNA. This prevents condensation by limiting opportunities to bridge to other molecules and stabilizes the RNA by locking it into a particular conformation. In contrast, for DNA, simulations suggest that spermine binds externally to the duplex, offering opportunities for intermolecular interaction. The goal of this study is to explain how RNA can remain soluble and available for interaction with other molecules in the cell despite the presence of spermine at concentrations high enough to precipitate DNA.