Molecular dynamics study of intrinsic stability in six RNA terminal loop motifs

Single stranded RNA molecules can assume a wide range of tertiary structures beyond the canonical A-form double helix. Certain sequences, termed motifs, are more common than a random distribution would suggest. The existence of such motifs can be rationalized in structural terms. In this study, we have investigated the intrinsic structural stability of RNA terminal loop motifs using multiple MD simulations in explicit water. Representative loops were chosen from the major tetraloop motifs, including also the U-turn motif. Not all loops retain their folded starting structure, but lowering the temperature to 277 K, or adding adjacent base pairs from the stem to which the motif is attached, helps stabilizing the folded loop structure.     

Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access.     

FR3D: finding local and composite recurrent structural motifs in RNA 3D structures

New methods are described for finding recurrent three-dimensional (3D) motifs in RNA atomic-resolution structures. Recurrent RNA 3D motifs are sets of RNA nucleotides with similar spatial arrangements. They can be local or composite. Local motifs comprise nucleotides that occur in the same hairpin or internal loop. Composite motifs comprise nucleotides belonging to three or more different RNA strand segments or molecules. We use a base-centered approach to construct efficient, yet exhaustive search procedures using geometric, symbolic, or mixed representations of RNA structure that we implement in a suite of MATLAB programs, “Find RNA 3D” (FR3D). The first modules of FR3D preprocess structure files to classify base-pair and -stacking interactions. Each base is represented geometrically by the position of its glycosidic nitrogen in 3D space and by the rotation matrix that describes its orientation with respect to a common frame. Base-pairing and base-stacking interactions are calculated from the base geometries and are represented symbolically according to the Leontis/Westhof basepairing classification, extended to include base-stacking. These data are stored and used to organize motif searches. For geometric searches, the user supplies the 3D structure of a query motif which FR3D uses to find and score geometrically similar candidate motifs, without regard to the sequential position of their nucleotides in the RNA chain or the identity of their bases. To score and rank candidate motifs, FR3D calculates a geometric discrepancy by rigidly rotating candidates to align optimally with the query motif and then comparing the relative orientations of the corresponding bases in the query and candidate motifs. Given the growing size of the RNA structure database, it is impossible to explicitly compute the discrepancy for all conceivable candidate motifs, even for motifs with less than ten nucleotides. The screening algorithm that we describe finds all candidate motifs whose geometric discrepancy with respect to the query motif falls below a user-specified cutoff discrepancy. This technique can be applied to RMSD searches. Candidate motifs identified geometrically may be further screened symbolically to identify those that contain particular basepair types or base-stacking arrangements or that conform to sequence continuity or nucleotide identity constraints. Purely symbolic searches for motifs containing user-defined sequence, continuity and interaction constraints have also been implemented. We demonstrate that FR3D finds all occurrences, both local and composite and with nucleotide substitutions, of sarcin/ricin and kink-turn motifs in the 23S and 5S ribosomal RNA 3D structures of the H. marismortui 50S ribosomal subunit and assigns the lowest discrepancy scores to bona fide examples of these motifs. The search algorithms have been optimized for speed to allow users to search the non-redundant RNA 3D structure database on a personal computer in a matter of minutes.     

Role of the closing base pair for d(GCA) hairpin stability: free energy analysis and folding simulations

Hairpin loops belong to the most important structural motifs in folded nucleic acids. The d(GNA) sequence in DNA can form very stable trinucleotide hairpin loops depending, however, strongly on the closing base pair. Replica-exchange molecular dynamics (REMD) were employed to study hairpin folding of two DNA sequences, d(gcGCAgc) and d(cgGCAcg), with the same central loop motif but different closing base pairs starting from single-stranded structures. In both cases, conformations of the most populated conformational cluster at the lowest temperature showed close agreement with available experimental structures. For the loop sequence with the less stable G:C closing base pair, an alternative loop topology accumulated as second most populated conformational state indicating a possible loop structural heterogeneity. Comparative-free energy simulations on induced loop unfolding indicated higher stability of the loop with a C:G closing base pair by ~3 kcal mol(-1) (compared to a G:C closing base pair) in very good agreement with experiment. The comparative energetic analysis of sampled unfolded, intermediate and folded conformational states identified electrostatic and packing interactions as the main contributions to the closing base pair dependence of the d(GCA) loop stability.     

The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs

The formation of genomic RNA dimers during the retroviral life cycle is essential for optimal viral replication and infectivity. The sequences and RNA structures responsible for this interaction are located in the untranslated 5' leader RNA, along with other cis-acting signals. Dimer formation occurs by specific interaction between identical structural motifs. It is believed that an initial kissing hairpin forms following self-recognition by autocomplementary RNA loops, leading to formation of an extended stable duplex. The dimerization initiation site (DIS) of the deltaretrovirus human T-cell lymphotropic virus type-I (HTLV-I) has been previously localized to a 14-nucleotide sequence predicted to contain an RNA stem loop. Biochemical probing of the monomeric RNA structure using RNAse T1, RNAse V1, RNAse U2, lead acetate, and dimethyl sulfate has led to the generation of the first structural map of the HTLV-I DIS. A comprehensive data set of individual nucleotide modifications reveals that the structural motif responsible for HTLV-I RNA dimerization forms a trinucleotide RNA loop, unlike any previously characterized retroviral dimerization motif. Molecular modeling demonstrates that this can be formed by an unusual C:synG base pair closing the loop. Comparative phylogeny indicates that such a motif may also exist in other deltaretroviruses.     

RNA aptamers that specifically bind to a 16S ribosomal RNA decoding region construct

RNA–RNA recognition is a critical process in controlling many key biological events, such as translation and ribozyme functions. The recognition process governing RNA–RNA interactions can involve complementary Watson–Crick (WC) base pair binding, or can involve binding through tertiary structural interaction. Hence, it is of interest to determine which of the RNA–RNA binding events might emerge through an in vitro selection process. The A-site of the 16S rRNA decoding region was chosen as the target, both because it possesses several different RNA structural motifs, and because it is the rRNA site where codon/anticodon recognition occurs requiring recognition of both mRNA and tRNA. It is shown here that a single family of RNA molecules can be readily selected from two different sizes of RNA library. The tightest binding aptamer to the A-site 16S rRNA construct, 109.2-3, has its consensus sequences confined to a stem–loop region, which contains three nucleotides complementary to three of the four nucleotides in the stem–loop region of the A-site 16S rRNA. Point mutations on each of the three nucleotides on the stem–loop of the aptamer abolish its binding capacity. These studies suggest that the RNA aptamer 109.2-3 interacts with the simple 27 nt A-site decoding region of 16S rRNA through their respective stem–loops. The most probable mode of interaction is through complementary WC base pairing, commonly referred to as a loop–loop ‘kissing’ motif. High affinity binding to the other structural motifs in the decoding region were not observed.     

The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn

The C/D guide RNAs predicted from the genomic sequences of three species of Pyrococcus delineate a family of small non-coding archaeal RNAs involved in the methylation of rRNA and tRNA. The C/D guides assemble into ribonucleoprotein (RNP) that contains the methyltransferase. The protein L7Ae, a key structural component of the RNP, binds to a Kink-turn (K-turn) formed by the C/D motif. The K-turn is a structure that consists of two RNA stems separated by a short asymmetric loop with a characteristic sharp bend (kink) between the two stems. The majority of the pyrococcal C/D guides contain a short 3 nt-spacer between the C′/D′ motifs. We show here that conserved terminal stem–loops formed by the C′/D′ motif of the Pyrococcus C/D RNAs are also L7Ae-binding sites. These stem–loops are related to the K-turn by sequence and structure, but they consist of a single stem closed by a terminal loop. We have named this structure the K-loop. We show that conserved non-canonical base pairs in the stem of the K-loop are necessary for L7Ae binding. For the C/D guides with a 3 nt-spacer we show that the sequence and length is also important. The K-loop could improve the stability of the C/D guide RNAs in Pyrococcal species, which are extreme hyperthermophiles.     

Clustering RNA structural motifs in ribosomal RNAs using secondary structural alignment

RNA structural motifs are the building blocks of the complex RNA architecture. Identification of non-coding RNA structural motifs is a critical step towards understanding of their structures and functionalities. In this article, we present a clustering approach for de novo RNA structural motif identification. We applied our approach on a data set containing 5S, 16S and 23S rRNAs and rediscovered many known motifs including GNRA tetraloop, kink-turn, C-loop, sarcin-ricin, reverse kink-turn, hook-turn, E-loop and tandem-sheared motifs, with higher accuracy than the state-of-the-art clustering method. We also identified a number of potential novel instances of GNRA tetraloop, kink-turn, sarcin-ricin and tandem-sheared motifs. More importantly, several novel structural motif families have been revealed by our clustering analysis. We identified a highly asymmetric bulge loop motif that resembles the rope sling. We also found an internal loop motif that can significantly increase the twist of the helix. Finally, we discovered a subfamily of hexaloop motif, which has significantly different geometry comparing to the currently known hexaloop motif. Our discoveries presented in this article have largely increased current knowledge of RNA structural motifs.     

Secondary structure model of the Mason-Pfizer monkey virus 5' leader sequence: identification of a structural motif common to a variety of retroviruses.

A stable secondary structure model is presented for the region 3' of the primer-binding site to 130 bases into the gag sequence of the prototype type D retrovirus Mason-Pfizer monkey virus. Using biochemical probing of RNA from this region in association with free energy minimization, we have identified a stem-loop structure in the region, which from other studies has been shown to be important for genomic RNA encapsidation. The structure involves a highly stable stem of five G-C pairs terminating in a heptaloop. Comparison of the Mason-Pfizer monkey virus structure with one predicted for squirrel monkey retrovirus demonstrates an identical stem and a common ACC motif in the loop. Free energy studies of the secondary structure of the 5' regions of eight other retroviruses predict stem loops which have similar GAYC motifs. We believe this may represent a common structural and sequence motif which among other functions may be involved in genomic RNA packaging in these viruses.     

The UA_handle: a versatile submotif in stable RNA architectures

Stable RNAs are modular and hierarchical 3D architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. Sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the UA_handle motif (5′XU/AN_nX3′). It consists of a U:A Watson–Crick: Hoogsteen trans base pair stacked over a classic Watson–Crick base pair, and a bulge of one or more nucleotides that can act as a handle for making different types of long-range interactions. This motif is one of the most versatile building blocks identified in stable RNAs. It enters into the composition of numerous recurrent motifs of greater structural complexity such as the T-loop, the 11-nt receptor, the UAA/GAN and the G-ribo motifs. Several structural principles pertaining to RNA motifs are derived from our analysis. A limited set of basic submotifs can account for the formation of most structural motifs uncovered in ribosomal and stable RNAs. Structural motifs can act as structural scaffoldings and be functionally and topologically equivalent despite sequence and structural differences. The sequence network resulting from the structural relationships shared by these RNA motifs can be used as a proto-language for assisting prediction and rational design of RNA tertiary structures.     

Characterization of the RNA motif responsible for the specific interaction of potato spindle tuber viroid RNA (PSTVd) and the tomato protein Virp1

Viroids are small non-coding parasitic RNAs that are able to infect their host plants systemically. This circular naked RNA makes use of host proteins to accomplish its proliferation. Here we analyze the specific binding of the tomato protein Virp1 to the terminal right domain of potato spindle tuber viroid RNA (PSTVd). We find that two asymmetric internal loops within the PSTVd (+) RNA, each composed of the sequence elements 5′-ACAGG and CUCUUCC-5′, are responsible for the specific RNA–protein interaction. In view of the nucleotide composition we call this structural element an ‘RY motif’. The RY motif located close to the terminal right hairpin loop of the PSTVd secondary structure has an ~5-fold stronger binding affinity than the more centrally located RY motif. Simultaneous sequence alterations in both RY motifs abolished the specific binding to Virp1. Mutations in any of the two RY motifs resulted in non-infectious viroid RNA, with the exception of one case, where reversion to sequence wild type took place. In contrast, the simultaneous exchange of two nucleotides within the terminal right hairpin loop of PSTVd had only moderate influence on the binding to Virp1. This variant was infectious and sequence changes were maintained in the progeny. The relevance of the phylogenetic conservation of the RY motif, and sequence elements therein, amongst various genera of the family Pospiviroidae is discussed.     

Effects of osmolytes on stable UUCG tetraloops and their preference for a CG closing base pair

Osmolytes have the potential to affect the stability of secondary structure motifs and alter preferences for conserved nucleic acid sequences in the cell. To contribute to the understanding of the in vivo function of RNA we observed the effects of different classes of osmolytes on the UNCG tetraloop motif. UNCG tetraloops are the most common and stable of the RNA tetraloops and are nucleation sites for RNA folding. They also have a significant thermodynamic preference for a CG closing base pair. The thermal denaturation of model hairpins containing UUCG loops was monitored using UV-Vis spectroscopy in the presence of osmolytes with different chemical properties. Interestingly, all of the osmolytes tested destabilized the hairpins, but all had little effect on the thermodynamic preference for a CG base pair, except for polyethylene glycol (PEG) 200. PEG 200 destabilized the loop with the CG closing base pair relative to the loop with a GC closing base pair. The destabilization was linear with increasing concentrations of PEG 200, and the slope of this relationship was not perturbed by changes in the hairpin stem outside of the closing pair. This result suggests that in the presence of PEG 200, the UUCG loop with a GC closing base pair may retain some preferential interactions with the cosolute that are lost in the presence of the CG closing base pair. These results reveal that relatively small structural changes may influence how osmolytes tune the stability, and thus the function of a secondary structure motif in vivo.     

RNA structural motif recognition based on least-squares distance

RNA structural motifs are recurrent structural elements occurring in RNA molecules. RNA structural motif recognition aims to find RNA substructures that are similar to a query motif, and it is important for RNA structure analysis and RNA function prediction. In view of this, we propose a new method known as RNA Structural Motif Recognition based on Least-Squares distance (LS-RSMR) to effectively recognize RNA structural motifs. A test set consisting of five types of RNA structural motifs occurring in Escherichia coli ribosomal RNA is compiled by us. Experiments are conducted for recognizing these five types of motifs. The experimental results fully reveal the superiority of the proposed LS-RSMR compared with four other state-of-the-art methods.     

Ni2+-binding RNA motifs with an asymmetric purine-rich internal loop and a G-A base pair.

RNA molecules with high affinity for immobilized Ni2+ were isolated from an RNA pool with 50 randomized positions by in vitro selection-amplification. The selected RNAs preferentially bind Ni2+ and Co2+ over other cations from first series transition metals. Conserved structure motifs, comprising about 15 nt, were identified that are likely to represent the Ni2+ binding sites. Two conserved motifs contain an asymmetric purine-rich internal loop and probably a mismatch G-A base pair. The structure of one of these motifs was studied with proton NMR spectroscopy and formation of the G-A pair at the junction of helix and internal loop was demonstrated. Using Ni2+ as a paramagnetic probe, a divalent metal ion binding site near this G-A base pair was identified. Ni2+ ions bound to this motif exert a specific stabilization effect. We propose that small asymmetric purine-rich loops that contain a G-A interaction may represent a divalent metal ion binding site in RNA.     

The common and the distinctive features of the bulged-G motif based on a 1.04 A resolution RNA structure

Bulged-G motifs are ubiquitous internal RNA loops that provide specific recognition sites for proteins and RNAs. To establish the common and distinctive features of the motif we determined the structures of three variants and compared them with related structures. The variants are 27-nt mimics of the sarcin/ricin loop (SRL) from Escherichia coli 23S ribosomal RNA that is an essential part of the binding site for elongation factors (EFs). The wild-type SRL has now been determined at 1.04 Å resolution, supplementing data obtained before at 1.11 Å and allowing the first calculation of coordinate error for an RNA motif. The other two structures, having a viable (C2658U^•G2663A) or a lethal mutation (C2658G^• G2663C), were determined at 1.75 and 2.25 Å resolution, respectively. Comparisons reveal that bulged-G motifs have a common hydration and geometry, with flexible junctions at flanking structural elements. Six conserved nucleotides preserve the fold of the motif; the remaining seven to nine vary in sequence and alter contacts in both grooves. Differences between accessible functional groups of the lethal mutation and those of the viable mutation and wild-type SRL may account for the impaired elongation factor binding to ribosomes with the C2658G^•G2663C mutation and may underlie the lethal phenotype.     

Evidence that folding of an RNA tetraloop hairpin is less cooperative than its DNA counterpart

Hairpin secondary structural elements play important roles in the folding and function of RNA and DNA molecules. Previous work from our lab on small DNA hairpin loop motifs, d(cGNAg) and d(cGNABg) (where B is C, G, or T), showed that folding is highly cooperative and obeys indirect coupling, consistent with a concerted transition. Herein, we investigate folding of the related, exceptionally stable RNA hairpin motif, r(cGNRAg) (where R is A or G). Previous NMR characterization identified a complex network of seven hydrogen bonds in this loop. We inserted three carbon (C3) spacers throughout the loop and found coupling between G1 of the loop and the CG closing base pair, similar to that found in DNA. These data support a GNRA motif being expandable at any position but before the G. Thermodynamic measurements of nucleotide-analogue-substituted oligonucleotides revealed pairwise-coupling free energies ranging from weak to strong. When coupling free energies were remeasured in the background of changes at a third site, they remained essentially unchanged even though all of the sites were coupled to each other. This type of coupling, referred to as "direct", is peculiar to the RNA loop. The data suggest that, for small stable loops, folding of RNA obeys a model with nearest-neighbor interactions, while folding of DNA follows a more concerted process in which the stabilizing interactions are linked through a conformational change. The lesser cooperativity in RNA loops may provide a more robust loop that can withstand mutations without a severe loss in stability. These differences may enhance the ability of RNA to evolve.     

A conserved lysine residue in the crenarchaea-specific loop is important for the crenarchaeal splicing endonuclease activity

In Archaea, splicing endonuclease (EndA) recognizes and cleaves precursor RNAs to remove introns. Currently, EndAs are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum. The endonuclease was classified into subfamily B, and the structure revealed that the enzyme lacks an N-terminal subdomain in the structural subunit. However, no structural information is available for crenarchaeal heterotetrameric EndAs that are predicted to belong to subfamily A. Here, we report the crystal structure of the EndA from Aeropyrum pernix, which is predicted to belong to subfamily A. The enzyme possesses the N-terminal subdomain in the structural subunit, revealing that the two subfamilies of heterotetrameric EndAs are structurally distinct. EndA from A. pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs. Our mutational study revealed that the conserved lysine residue in the loop is important for endonuclease activity. Furthermore, the sequence characteristics of the loops and the positions towards the substrate RNA according to a docking model prompted us to propose that crenarchaea-specific loops and an extra amino acid sequence at the catalytic loop of nanoarchaeal EndA are derived by independent convergent evolution and function for recognizing noncanonical bulge-helix-bulge motif RNAs as substrates.     

Solution Structure of a GAAG Tetraloop in Helix 6 of SRP RNA from Pyrococcus furiosus

The NMR structure of a 12-mer RNA derived from the helix 6 of SRP RNA from Pyrococcus furiosus, whose loop-closing base pair is U:G, was determined, and the structural and thermodynamic properties of the RNA were compared with those of a mutant RNA with the C:G closing base pair. Although the structures of the two RNAs are similar to each other and adopt the GNRR motif, the conformational stabilities are significantly different to each other. It was suggested that weaker stacking interaction of the GAAG loop with the U:G closing base pair in 12-mer RNA causes the lower conformational stability.     

Promoting RNA helical stacking via A-minor junctions

RNA molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3D architectures. The A-minor junction is a class of RNA motifs that specifically controls coaxial stacking of helices in natural RNAs. A sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified A-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. This class of modular motifs follows a topological rule that can accommodate a variety of interchangeable A-minor interactions with distinct local structural motifs. Overall, two different types of A-minor junctions can be distinguished based on their functional self-assembling behavior: one group makes use of triloops or GNRA and GNRA-like loops assembling with helices, while the other takes advantage of more complex tertiary receptors specific for the loop to gain higher stability. This study demonstrates how different structural motifs of RNA can contribute to the formation of topologically equivalent helical stacks. It also exemplifies the need of classifying RNA motifs based on their tertiary structural features rather than secondary structural features. The A-minor junction rule can be used to facilitate tertiary structure prediction of RNAs and rational design of RNA parts for nanobiotechnology and synthetic biology.     

A comparative study on two GNRA-tetraloop receptors: 11-nt and IC3 motifs

Natural RNAs often contain terminal loops consisting of GNRA (N=A, G, C, U; R=A, G) and their receptors, which bind to the loops via long-range RNA-RNA interactions. Among several known receptors, two characteristic structural elements have been identified that are termed the 11-nt motif (CCUAAG-UAUGG) and IC3 motif (CCCUAAC-GAGGG). These two motifs that share a similar secondary structure have been shown to exhibit distinctively different binding specificities. The 11-nt motif recognizes a GAAA loop with highest specificity among the known receptors, whereas the IC3 motif distinguishes GAAA from other GNRA loops less stringently than any other receptors. To identify the elements in the receptors that determine the binding specificity, a series of chimeric receptors derived from the two motifs were prepared and their properties were examined. We identified characteristic base-pairs and a particular U residue in the receptors as such elements by means of a gel mobility shift assay that evaluates the degree of the tetraloop-receptor interaction. The relationship between the elements and the specificity is discussed together with a model that describes a possible evolutional linkage between the two receptors.