RNA structure: experimental analysis

Among all of the biological macromolecules, the functional versatility of RNAs is unique including encoding or transferring genetic information and performing catalysis. These biological functions are highly dependent upon RNA folding and structure. Since the discovery of catalytic RNAs in the early 1980s, a recent breakthrough came from the identification of a wealth of micro RNAs, small interfering RNAs and regulatory RNAs, all involved in modulation of gene expression. The structure of these novel RNAs, either free or in complex with specific ligands, can be analyzed using various experimental strategies, including X-ray crystallography, cryo-electron microscopy, nuclear magnetic resonance spectroscopy, structure-specific probes, with some that can be used in living cells, RNA engineering, thermal denaturation and mass spectrometry. Among these, X-ray crystallography has recently enabled determination of the structures of several large and complex RNAs, as well as of ribonucleoprotein complexes. The database of RNA structure has grown tremendously since the recent crystal structure analyses of the prokaryotic ribosome and its subunits. These methods are now widely applied to a variety of biologically relevant RNAs.     

RNA crystallization

RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to synthesize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.     

Three-dimensional RNA structure refinement by hydroxyl radical probing

Molecular modeling guided by experimentally derived structural information is an attractive approach for three-dimensional structure determination of complex RNAs that are not amenable to study by high-resolution methods. Hydroxyl radical probing (HRP), which is performed routinely in many laboratories, provides a measure of solvent accessibility at individual nucleotides. HRP measurements have, to date, only been used to evaluate RNA models qualitatively. Here we report the development of a quantitative structure refinement approach using HRP measurements to drive discrete molecular dynamics simulations for RNAs ranging in size from 80 to 230 nucleotides. We first used HRP reactivities to identify RNAs that form extensive helical packing interactions. For these RNAs, we achieved highly significant structure predictions given the inputs of RNA sequence and base pairing. This HRP-directed tertiary structure refinement approach generates robust structural hypotheses that are useful for guiding explorations of structure-function inter-relationships in RNA.     

Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module

Well-ordered crystals of a genomic hepatitis delta virus (HDV) ribozyme, a large, globular RNA, were obtained employing a new crystallization method. A high-affinity binding site for the spliceosomal protein U1A was engineered into a segment of the catalytic RNA that is dispensable for catalysis. Because molecular surfaces of proteins are more chemically varied than those of RNA, the presence of the protein moiety was expected to facilitate crystallization and improve crystal order. The HDV ribozyme-U1A complex crystallized readily, and its structure was solved using standard techniques for heavy-atom derivatization of protein crystals. Over 1200 A(2) of the solvent-accessible surface area of the complex are involved in crystal contacts. As protein-protein interactions comprise 85% of this buried area, these crystals appear to be held together predominantly by the protein component of the complex. Our crystallization method should be useful for the structure determination of other biochemically important RNAs for which protein partners do not exist or are experimentally intractable. The refined model of the complex (R-free=27.9% for all reflections between 20.0 and 2.3 A) reveals an RNA with a deep active site cleft. Well-ordered metal ions are not observed crystallographically in this cavity. Biochemical results of previous workers had suggested an important role in catalysis for cytosine 75. The pyrimidine base of this residue is buried at the bottom of the active site in an environment that could raise its pK(a) value. We propose that this highly conserved cytosine may be the general base that catalyzes the transesterification.     

Microarrays for identifying binding sites and probing structure of RNAs

Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure–function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs.     

Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination

Counterdiffusion crystallization in capillary is a very simple, cost-effective, and practical procedure for obtaining protein crystals suitable for X-ray data analysis. Its principles have been derived using well-known concepts coupling the ideas of precipitation and diffusion mass transport in a restricted geometry. The counterdiffusion process has been used to simultaneously screen for optimal conditions for protein crystal growth, incorporate strong anomalous scattering atoms, and mix in cryogenic solutions in a single capillary tube. The crystals obtained in the capillary have been used in situ for X-ray analysis. The implementation of this technique linked to the advancement of current crystallography software leads to a powerful structure determination method consolidating crystal growth, X-ray data collection, and ab initio phase determination into one without crystal manipulation. We review the historical progress of counterdiffusion crystallization, its application to X-ray crystallography, and ongoing tool development for high-throughput protein structure determination.     

Determining RNA three-dimensional structures using low-resolution data

Knowing the 3-D structure of an RNA is fundamental to understand its biological function. Nowadays X-ray crystallography and NMR spectroscopy are systematically applied to newly discovered RNAs. However, the application of these high-resolution techniques is not always possible, and thus scientists must turn to lower resolution alternatives. Here, we introduce a pipeline to systematically generate atomic resolution 3-D structures that are consistent with low-resolution data sets. We compare and evaluate the discriminative power of a number of low-resolution experimental techniques to reproduce the structure of the Escherichia coli tRNA(VAL) and P4-P6 domain of the Tetrahymena thermophila group I intron. We test single and combinations of the most accessible low-resolution techniques, i.e. hydroxyl radical footprinting (OH), methidiumpropyl-EDTA (MPE), multiplexed hydroxyl radical cleavage (MOHCA), and small-angle X-ray scattering (SAXS). We show that OH-derived constraints are accurate to discriminate structures at the atomic level, whereas EDTA-based constraints apply to global shape determination. We provide a guide for choosing which experimental techniques or combination of thereof is best in which context. The pipeline represents an important step towards high-throughput low-resolution RNA structure determination.     

限制性酶切提高E.coli Hfq蛋白晶体结构分辨率

Hfq蛋白是一种促进sRNA结合互补RNA的细菌伴侣蛋白. Hfq蛋白可以抑制或上调目的蛋白的表达及促进mRNA的降解,从而调控细菌的生理功能使其适应周围胁迫环境.Hfq是同源六聚体蛋白质,在其中心部位有1个内环,每个单体上有1个极其保守的组氨酸分布在这个内环表面.根据Hfq这个特点,应用镍离子亲和柱直接从大肠杆菌中纯化Hfq,接着用凝胶过滤层析柱纯化,最终获得了纯度70%的Hfq蛋白.对该蛋白质进行结晶条件初筛前,应用胰凝乳蛋白酶对其进行限制性蛋白酶解,切除其C端无规则卷曲,便于Hfq结晶及提高晶体衍射率.初期得到的Hfq晶体是极小的针状晶体,通过优化结晶条件获得了较大的晶体.最终解析出了2个分辨率较高的Hfq晶体结构,其中1个晶体结构分辨率达到了1.63A.     

Reconstruction of Natural RNA Sequences from RNA Shape,Thermodynamic Stability,Mutational Robustness,and Linguistic Complexity by Evolutionary Computation

Abstract

The process of designing novel RNA sequences by inverse RNA folding, available in tools such as RNAinverse and InfoRNA, can be thought of as a reconstruction of RNAs from secondary structure. In this reconstruction problem, no physical measures are considered as additional constraints that are independent of structure, aside of the goal to reach the same secondary structure as the input using energy minimization methods. An extension of the reconstruction problem can be formulated since in many cases of natural RNAs, it is desired to analyze the sequence and structure of RNA molecules using various physical quantifiable measures. In prior works that used secondary structure predictions, it has been shown that natural RNAs differ significantly from random RNAs in some of these measures. Thus, we relax the problem of reconstructing RNAs from secondary structure into reconstructing RNAs from shapes, and in turn incorporate physical quantities as constraints. This allows for the design of novel RNA sequences by inverse folding while considering various physical quantities of interest such as thermodynamic stability, mutational robustness, and linguistic complexity. At the expense of altering the number of nucleotides in stems and loops, for example, physical measures can be taken into account. We use evolutionary computation for the new reconstruction problem and illustrate the procedure on various natural RNAs.     

Reconstruction of natural RNA sequences from RNA shape, thermodynamic stability, mutational robustness, and linguistic complexity by evolutionary computation

The process of designing novel RNA sequences by inverse RNA folding, available in tools such as RNAinverse and InfoRNA, can be thought of as a reconstruction of RNAs from secondary structure. In this reconstruction problem, no physical measures are considered as additional constraints that are independent of structure, aside of the goal to reach the same secondary structure as the input using energy minimization methods. An extension of the reconstruction problem can be formulated since in many cases of natural RNAs, it is desired to analyze the sequence and structure of RNA molecules using various physical quantifiable measures. In prior works that used secondary structure predictions, it has been shown that natural RNAs differ significantly from random RNAs in some of these measures. Thus, we relax the problem of reconstructing RNAs from secondary structure into reconstructing RNAs from shapes, and in turn incorporate physical quantities as constraints. This allows for the design of novel RNA sequences by inverse folding while considering various physical quantities of interest such as thermodynamic stability, mutational robustness, and linguistic complexity. At the expense of altering the number of nucleotides in stems and loops, for example, physical measures can be taken into account. We use evolutionary computation for the new reconstruction problem and illustrate the procedure on various natural RNAs.     

The three-dimensional folding of the tRNA-like structure of tobacco mosaic virus RNA. A new building principle applied twice

The structure of the tRNA-like 3' terminus of tobacco mosaic virus (TMV) RNA has been studied. A 3' -terminal fragment possessing the tRNA-like properties was probed with chemical modification and enzymatic digestions. A model of the secondary structure is proposed for the last 105 nucleotides. The corresponding region of other tobamoviral RNAs can be folded in an identical secondary structure. A three-dimensional model for the tRNA-like structure is given which is compared with those proposed earlier for the tRNA-like 3' termini of turnip yellow mosaic virus (TYMV) RNA and brome mosaic virus (BMV) RNA. A new building principle which we discovered previously by studying the latter RNAs appears to be applied twice in the tRNA-like structure of TMV RNA. The determination of the minimal length requirement for recognition of CTP, ATP:tRNA nucleotidyl-transferase reveals a size of ˜100 nucleotides in agreement with the models proposed.     

Solution structure of RNase P RNA

The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.     

Strategies for crystallization and structure determination of very large macromolecular assemblies

High-resolution structures of macromolecular assemblies are pivotal for our understanding of their biological functions in fundamental cellular processes. In the field of X-ray crystallography, recent methodological and instrumental advances have led to the structure determinations of macromolecular assemblies of increased size and complexity, such as those of ribosomal complexes, RNA polymerases, and large multifunctional enzymes. These advances include the use of robotic screening techniques that maximize the chances of obtaining well-diffracting crystals of large complexes through the fine sampling of crystallization space. Sophisticated crystal optimization and cryoprotection techniques and the use of highly brilliant X-ray beams from third-generation synchrotron light sources now allow data collection from weakly diffracting crystals with large asymmetric units. Combined approaches are used to derive phase information, including phases calculated from electron microscopy (EM) models, heavy atom clusters, and density modification protocols. New crystallographic software tools prove valuable for structure determination and model refinement of large macromolecular complexes.     

In vitro selections with RNAs of variable length converge on a robust catalytic core

In vitro selection is a powerful tool that can be used to understand basic principles of molecular evolution. We used in vitro selection to understand how changes in length and the accumulation of point mutations enable the evolution of functional RNAs. Using RNA populations of various lengths, we performed a series of in vitro experiments to select for ribozymes with RNA ligase activity. We identified a core ribozyme structure that was robust to changes in RNA length, high levels of mutagenesis, and increased selection pressure. Elaboration on this core structure resulted in improved activity which we show is consistent with a larger trend among functional RNAs in which increasing motif size can lead to an exponential improvement in fitness. We conclude that elaboration on conserved core structures is a preferred mechanism in RNA evolution. This conclusion, drawn from selections of RNAs from random sequences, is consistent with proposed evolutionary histories of specific biological RNAs. More generally, our results indicate that modern RNA structures can be used to infer ancestral structures. Our observations also suggest a mechanism by which structural outcomes of early RNA evolution would be largely reproducible even though RNA fitness landscapes consist of disconnected clusters of functional sequences.     

An integrative NMR-SAXS approach for structural determination of large RNAs defines the substrate-free state of a trans-cleaving Neurospora Varkud Satellite ribozyme

The divide-and-conquer strategy is commonly used for protein structure determination, but its applications to high-resolution structure determination of RNAs have been limited. Here, we introduce an integrative approach based on the divide-and-conquer strategy that was undertaken to determine the solution structure of an RNA model system, the Neurospora VS ribozyme. NMR and SAXS studies were conducted on a minimal trans VS ribozyme as well as several isolated subdomains. A multi-step procedure was used for structure determination that first involved pairing refined NMR structures with SAXS data to obtain structural subensembles of the various subdomains. These subdomain structures were then assembled to build a large set of structural models of the ribozyme, which was subsequently filtered using SAXS data. The resulting NMR-SAXS structural ensemble shares several similarities with the reported crystal structures of the VS ribozyme. However, a local structural difference is observed that affects the global fold by shifting the relative orientation of the two three-way junctions. Thus, this finding highlights a global conformational change associated with substrate binding in the VS ribozyme that is likely critical for its enzymatic activity. Structural studies of other large RNAs should benefit from similar integrative approaches that allow conformational sampling of assembled fragments.