Jason R. Stagno

Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.

Riboswitches are structural RNA elements that are generally located in the 5′ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

Synthesis and applications of RNAs with position-selective labelling and mosaic composition

Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid–liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch for nuclear magnetic resonance spectroscopy or single-molecule Förster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection, and disease diagnostics.

Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination

Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusB–NusE–BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB–NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein–protein and protein–RNA interactions. Further crystallographic investigation of a NusB–NusE–dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex.

Small-angle X-ray scattering: a bridge between RNA secondary structures and three-dimensional topological structures

Whereas the structures of small to medium-sized well folded RNA molecules often can be determined by either X-ray crystallography or NMR spectroscopy, obtaining structural information for large RNAs using experimental, computational, or combined approaches remains a major interest and challenge. RNA is very sensitive to small-angle X-ray scattering (SAXS) due to high electron density along phosphate-sugar backbones, whose scattering contribution dominates SAXS intensity. For this reason, SAXS is particularly useful in obtaining global RNA structural information that outlines backbone topologies and, therefore, molecular envelopes. Such information is extremely valuable in bridging the gap between the secondary structures and three-dimensional topological structures of RNA molecules, particularly those that have proven difficult to study using other structure-determination methods. Here we review published results of RNA topological structures derived from SAXS data or in combination with other experimental data, as well as details on RNA sample preparation for SAXS experiments.

Applications of PLOR in labeling large RNAs at specific sites.

Incorporation of modified or labeled nucleotides at specific sites in RNAs is critical for gaining insights into the structure and function of RNAs. Preparation of site-specifically labeled large RNAs in amounts suitable for structural or functional studies is extremely difficult using current methodologies. The position-selective labeling of RNA, PLOR, is a recently developed method that makes such syntheses possible. PLOR allows incorporation of various probes, including (2)D/(13)C/(15)N-isotopic labels, Cy3/Cy5/Alexa488/Alexa555 fluorescent dyes, biotin and other chemical groups, into specific positions in long RNAs. Here, we describe in detail the use of PLOR to label RNAs at specific segment(s) or discrete sites.

Modeling RNA topological structures using small angle X-ray scattering

Detailed understanding of the structure and function relationship of RNA requires knowledge about RNA three-dimensional (3D) topological folding. However, there are very few unique RNA entries in structure databases. This is due to challenges in determining 3D structures of RNA using conventional methods, such as X-ray crystallography and NMR spectroscopy, despite significant advances in both of these technologies. Computational methods have come a long way in accurately predicting the 3D structures of small (<50nt) RNAs to within a few angstroms compared to their native folds. However, lack of an apparent correlation between an RNA primary sequence and its 3D fold ultimately limits the success of purely computational approaches. In this context, small angle X-ray scattering (SAXS) serves as a valuable tool by providing global shape information of RNA. In this article, we review the progress in determining RNA 3D topological structures, including a new method that combines secondary structural information and SAXS data to sample conformations generated through hierarchical moves of commonly observed RNA motifs.

Real‐time crystallographic studies of the adenine riboswitch using an X‐ray free‐electron laser

Structures of the four reaction states of the adenine riboswitch aptamer domain, including a transient intermediate state were solved by serial femtosecond crystallography. The structures not only demonstrate the use of X‐ray free‐electron lasers for RNA crystallography but have also proven that transient states can be determined in real time by mix‐and‐inject crystallography. These results illustrate the structural basis for the ligand‐induced conformational changes associated with the molecular ‘switch’.

Crystal structure of a plectonemic RNA supercoil

Genome packaging is an essential housekeeping process in virtually all organisms for proper storage and maintenance of genetic information. Although the extent and mechanisms of packaging vary, the process involves the formation of nucleic-acid superstructures. Crystal structures of DNA coiled coils indicate that their geometries can vary according to sequence and/or the presence of stabilizers such as proteins or small molecules. However, such superstructures have not been revealed for RNA. Here we report the crystal structure of an RNA supercoil, which displays one level higher molecular organization than previously reported structures of DNA coiled coils. In the presence of an RNA-binding protein, two interlocking RNA coiled coils of double-stranded RNA, a ‘coil of coiled coils’, form a plectonemic supercoil. Molecular dynamics simulations suggest that protein-RNA interaction is required for the stability of the supercoiled RNA. This study provides structural insight into higher-order packaging mechanisms of nucleic acids.

Topological Structure Determination of RNA Using Small-Angle X-Ray Scattering

Knowledge of RNA three-dimensional topological structures provides important insight into the relationship between RNA structural components and function. It is often likely that near-complete sets of biochemical and biophysical data containing structural restraints are not available, but one still wants to obtain knowledge about approximate topological folding of RNA. In this regard, general methods for determining such topological structures with minimum readily available restraints are lacking. Naked RNAs are difficult to crystallize and NMR spectroscopy is generally limited to small RNA fragments. By nature, sequence determines structure and all interactions that drive folding are self-contained within sequence. Nevertheless, there is little apparent correlation between primary sequences and three-dimensional folding unless supplemented with experimental or phylogenetic data. Thus, there is an acute need for a robust high-throughput method that can rapidly determine topological structures of RNAs guided by some experimental data. We present here a novel method (RS3D) that can assimilate the RNA secondary structure information, small-angle X-ray scattering data, and any readily available tertiary contact information to determine the topological fold of RNA. Conformations are firstly sampled at glob level where each glob represents a nucleotide. Best-ranked glob models can be further refined against solvent accessibility data, if available, and then converted to explicit all-atom coordinates for refinement against SAXS data using the Xplor-NIH program. RS3D is widely applicable to a variety of RNA folding architectures currently present in the structure database. Furthermore, we demonstrate applicability and feasibility of the program to derive low-resolution topological structures of relatively large multi-domain RNAs.

Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA

Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13C15N-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.