Analysis of Four-Way Junctions in RNA Structures

RNA secondary structures can be divided into helical regions composed of canonical Watson-Crick and related base pairs, as well as single-stranded regions such as hairpin loops, internal loops, and junctions. These elements function as building blocks in the design of diverse RNA molecules with various fundamental functions in the cell. To better understand the intricate architecture of three-dimensional (3D) RNAs, we analyze existing RNA four-way junctions in terms of base-pair interactions and 3D configurations. Specifically, we identify nine broad junction families according to coaxial stacking patterns and helical configurations. We find that helices within junctions tend to arrange in roughly parallel and perpendicular patterns and stabilize their conformations using common tertiary motifs such as coaxial stacking, loop-helix interaction, and helix packing interaction. Our analysis also reveals a number of highly conserved base-pair interaction patterns and novel tertiary motifs such as A-minor-coaxial stacking combinations and sarcin/ricin motif variants. Such analyses of RNA building blocks can ultimately help in the difficult task of RNA 3D structure prediction.     

Tertiary interactions between helices h13 and h44 in 16S RNA contribute to the fidelity of translation

The A-minor interaction, formed between single-stranded adenosines and the minor groove of a receptor helix, is among the most common motifs found in rRNA. Among the A-minors found in 16S rRNA are a set of interactions between adenosines at positions 1433, 1434 and 1468 in helix 44 (h44) and their receptors in the nucleotide 320-340 region of helix 13 (h13). These interactions have been implicated in the maintenance of translational accuracy, because base substitutions at the adjacent C1469 increase miscoding errors. We have tested their functional significance through mutagenesis of h13 and h44. Mutations at the h44 A residues, or the A-minor receptors in h13, increase a variety of translational errors and a subset of the mutants show decreased association between 30S and 50S ribosomal subunits. These results are consistent with the involvement of h13-h44 interactions in the alignment and packing of these helices in the 30S subunit and the importance of this helical alignment for tRNA selection and subunit-subunit interaction.     

RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex

Tertiary interactions are critical for proper RNA folding and ribozyme catalysis. RNA tertiary structure is often condensed through long-range helical packing interactions mediated by loop-receptor motifs. RNA structures displaying helical packing by loop-receptor interactions have been solved by X-ray crystallography, but not by NMR. Here, we report the NMR structure of a 30 kDa GAAA tetraloop-receptor RNA complex. In order to stabilize the complex, we used a modular design in which the RNA was engineered to form a homodimer, with each subunit containing a GAAA tetraloop phased one helical turn apart from its cognate 11-nucleotide receptor domain. The structure determination utilized specific isotopic labeling patterns (2H, 13C and 15N) and refinement against residual dipolar couplings. We observe a unique and highly unusual chemical shift pattern for an adenosine platform interaction that reveals a spectroscopic fingerprint for this motif. The structure of the GAAA tetraloop-receptor interaction is well defined solely from experimental NMR data, shows minor deviations from previously solved crystal structures, and verifies the previously inferred hydrogen bonding patterns within this motif. This work demonstrates the feasibility of using engineered homodimers as modular systems for the determination of RNA tertiary interactions by NMR.     

An innate twist between Crick’s wobble and Watson-Crick base pairs

Non-Watson-Crick pairs like the G·U wobble are frequent in RNA duplexes. Their geometric dissimilarity (nonisostericity) with the Watson-Crick base pairs and among themselves imparts structural variations decisive for biological functions. Through a novel circular representation of base pairs, a simple and general metric scheme for quantification of base-pair nonisostericity, in terms of residual twist and radial difference that can also envisage its mechanistic effect, is proposed. The scheme is exemplified by G·U and U·G wobble pairs, and their predicable local effects on helical twist angle are validated by MD simulations. New insights into a possible rationale for contextual occurrence of G·U and other non-WC pairs, as well as the influence of a G·U pair on other non-Watson-Crick pair neighborhood and RNA-protein interactions are obtained from analysis of crystal structure data. A few instances of RNA-protein interactions along the major groove are documented in addition to the well-recognized interaction of the G·U pair along the minor groove. The nonisostericity-mediated influence of wobble pairs for facilitating helical packing through long-range interactions in ribosomal RNAs is also reviewed.     

Hydrogen and hydration of DNA and RNA oligonucleotides

In this paper, hydrogen bonding interaction and hydration in crystal structures of both DNA and RNA oligonucleotides are discussed. Their roles in the formation and stabilization of oligonucleotides have been covered. Details of the Watson-Crick base pairs G.C and A.U in DNA and RNA are illustrated. The geometry of the wobble (mismatched) G.U base pairs and the cis and almost trans conformations of the mismatched U.U base pairs in RNA is described. The difference in hydration of the Watson-Crick base pairs G.C, A.U and the wobble G.U in different sequences of codon-anticodon interaction in double helical molecules are indicative of the effect of hydration. The hydration patterns of the phosphate, the 2'-hydroxyl groups, the water bridges linking the phosphate group, N7 (purine) and N4 of Cs or O4 of Us in the major groove, the water bridges between the 2'-hydroxyl group and N3 (purine) and O2 (pyrimidine) in the minor groove are discussed.     

Structure, dynamics and hydration of the nogalamycin-d(ATGCAT)2Complex determined by NMR and molecular dynamics simulations in solution.

The structure of the 1:1 nogalamycin:d(ATGCAT)2 complex has been determined in solution from high-resolution NMR data and restrained molecular dynamics (rMD) simulations using an explicit solvation model. The antibiotic intercalates at the 5'-TpG step with the nogalose lying along the minor groove towards the centre of the duplex. Many drug-DNA nuclear Overhauser enhancements (NOEs) in the minor groove are indicative of hydrophobic interactions over the TGCA sequence. Steric occlusion prevents a second nogalamycin molecule from binding at the symmetry-related 5'-CpA site, leading to the conclusion that the observed binding orientation in this complex is the preferred orientation free of the complication of end-effects (drug molecules occupy terminal intercalation sites in all X-ray structures) or steric interactions between drug molecules (other NMR structures have two drug molecules bound in close proximity), as previously suggested. Fluctuations in key structural parameters such as rise, helical twist, slide, shift, buckle and sugar pucker have been examined from an analysis of the final 500 ps of a 1 ns rMD simulation, and reveal that many sequence-dependent structural features previously identified by comparison of different X-ray structures lie within the range of dynamic fluctuations observed in the MD simulations. Water density calculations on MD simulation data reveal a time-averaged pattern of hydration in both the major and minor groove, in good agreement with the extensive hydration observed in two related X-ray structures in which nogalamycin is bound at terminal 5'-TpG sites. However, the pattern of hydration determined from the sign and magnitude of NOE and ROE cross-peaks to water identified in 2D NOESY and ROESY experiments identifies only a few "bound" water molecules with long residence times. These solvate the charged bicycloaminoglucose sugar ring, suggesting an important role for water molecules in mediating drug-DNA electrostatic interactions within the major groove. The high density of water molecules found in the minor groove in X-ray structures and MD simulations is found to be associated with only weakly bound solvent in solution.     

Tertiary Motifs Revealed in Analyses of Higher-Order RNA Junctions

RNA junctions are secondary-structure elements formed when three or more helices come together. They are present in diverse RNA molecules with various fundamental functions in the cell. To better understand the intricate architecture of three-dimensional (3D) RNAs, we analyze currently solved 3D RNA junctions in terms of base-pair interactions and 3D configurations. First, we study base-pair interaction diagrams for solved RNA junctions with 5 to 10 helices and discuss common features. Second, we compare these higher-order junctions to those containing 3 or 4 helices and identify global motif patterns such as coaxial stacking and parallel and perpendicular helical configurations. These analyses show that higher-order junctions organize their helical components in parallel and helical configurations similar to lower-order junctions. Their sub-junctions also resemble local helical configurations found in three- and four-way junctions and are stabilized by similar long-range interaction preferences such as A-minor interactions. Furthermore, loop regions within junctions are high in adenine but low in cytosine, and in agreement with previous studies, we suggest that coaxial stacking between helices likely forms when the common single-stranded loop is small in size; however, other factors such as stacking interactions involving noncanonical base pairs and proteins can greatly determine or disrupt coaxial stacking. Finally, we introduce the ribo-base interactions: when combined with the along-groove packing motif, these ribo-base interactions form novel motifs involved in perpendicular helix-helix interactions. Overall, these analyses suggest recurrent tertiary motifs that stabilize junction architecture, pack helices, and help form helical configurations that occur as sub-elements of larger junction networks. The frequent occurrence of similar helical motifs suggest nature's finite and perhaps limited repertoire of RNA helical conformation preferences. More generally, studies of RNA junctions and tertiary building blocks can ultimately help in the difficult task of RNA 3D structure prediction.     

Crystal and molecular structure of the A-DNA dodecamer d(CCGTACGTACGG). Choice of fragment helical axis.

The crystal structure of the dodecamer d(CCGTACGTACGG) has been determined at 2.5 A resolution. The crystals grow in the hexagonal space group P6(1)22, a = b = 46.2 A, c = 71.5 A with one strand as the asymmetric unit. Diffraction data were collected by the oscillation film method yielding 1664 unique reflections with an Rmerge of 0.04. The structure was solved by real-space rotational translational searches with idealized helical models of A, B and Z-DNA. The best agreement was given by an A-DNA model with its dyad axis along the diagonal crystallographic dyad axis, with an R-factor 0.43 and correlation coefficient of 0.59 for data between 10 and 5 A. Iterative map fitting and restrained least-squares refinement and addition of 40 solvent molecules brought the R-factor to 0.15 and the correlation coefficient to 0.97 for all data between 8.0 and 2.5 A. The stereochemistry of the atomic model is good, with a root-mean-square deviation in bond distances of 0.006 A. This is the first example of an A-DNA containing a full helical turn. The dodecamer displays a novel packing motif. In addition to the characteristic contacts between the terminal base-pairs and the minor grooves of symmetry-related molecules, there are also minor groove to minor groove interactions not previously observed. The packing leaves an approximately 25 A diameter solvent channel around the origin, along the c-axis. The presence of a prominent 3.4 A meridional reflection and other diffuse features in the diffraction pattern provided evidence for the presence of disordered B-DNA along the c-axis, which can be accommodated in these solvent channels. The molecular conformation of the dodecamer also displays novel features. The dyad-related halves of the molecule are bent at an angle of 20 degrees, and the helical parameters are affected by this bend. Unlike the shorter A-DNA octamers, the dimensions of the major groove can be directly measured. Novel correlations between local helical parameters and global conformational features are presented. Most of the solvent molecules are associated with the major groove and the sugar-phosphate backbone.     

Intrinsic bending and deformability at the T-A step of CCTTTAAAGG: a comparative analysis of T-A and A-T steps within A-tracts

Introduction of a T-A or pyrimidine-purine step into a straight and rigid A-tract can cause a positive roll deformation that kinks the DNA helix at that step. In CCTTTAAAGG, the central T-A step has an 8.6 degrees bend toward the major groove. We report the structural analysis of CCTTTAAAGG and a comparison with 25 other representative crystal structures from the NDB containing at least four consecutive A or T bases. On average, more local bending occurs at the disruptive T-A step (8.21 degrees ) than at an A-T step (5.71 degrees ). In addition, A-tracts containing an A-T step are more bent than are pure A-tracts, and hence A-A and A-T steps are not equivalent. All T-A steps examined exhibit positive roll, bending towards the major groove, while A-T steps display negative roll and bend slightly towards the minor groove. This illustrates how inherent negative and positive roll are, respectively, at A-T and T-A steps within A-tracts. T-A steps are more deformable, showing larger and more variable deformations of minor groove width, rise, cup, twist, and buckle. Standard deviations of twist, rise, and cup for T-A steps are 6.66 degrees, 0.55 A, and 15.90 degrees, versus 2.28 degrees, 0.21 A, and 2.99 degrees for A-T steps. Packing constraints determine which local values of these helical parameters an individual T-A step will adopt. For instance, with CCTTTAAAGG and three isomorphous structures, CGATTAATCG, CGATATATCG, and CGATCGATCG, crystal packing forces lead to a series of correlated changes: widened minor groove, large slide, low twist, and large rise. The difference in helical parameters between A-T steps lying within A-tracts, versus A-T steps within alternating AT sequences, demonstrates the importance of neighboring steps on the conformation of a given dinucleotide step.     

Crystal structure of 2'-O-Me(CGCGCG)2, an RNA duplex at 1.30 A resolution. Hydration pattern of 2'-O-methylated RNA.

The molecular and crystal structure of 2'-O-Me (CGCGCG)2 has been determined using synchrotron radiation at near-atomic resolution (1.30 A), the highest resolution to date in the RNA field. The crystal structure is a half-turn A-type helix with some helical parameters deviating from canonical A-RNA, such as low base pair rise, elevated helical twist and inclination angles. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. This type of regularity is not seen in other RNA crystal structures. The structure includes 44 water molecules and two hydrated Mg2+ions one of which lies exactly on the crystallographic 2-fold axis. There are distinct patterns of hydration in the major and the minor grooves. The major groove is stabilised by water clusters consisting of fused five- and six-membered rings. Minor groove contains only a single row of water molecules; each water bridges either two self-parallel cytosines or two self-parallel guanines by a pair of hydrogen bonds. The structure provides the first view of the hydration scheme of 2'-O-methylated RNA duplex.     

Crystal structure of a model branchpoint-U2 snRNA duplex containing bulged adenosines

Bulged nucleotides play a variety of important roles in RNA structure and function, frequently forming tertiary interactions and sometimes even participating in RNA catalysis. In pre-mRNA splicing, the U2 snRNA base pairs with the intron branchpoint sequence (BPS) to form a short RNA duplex that contains a bulged adenosine that ultimately serves as the nucleophile that attacks the 5' splice site. We have determined a 2.18-A resolution crystal structure of a self-complementary RNA designed to mimic the highly conserved yeast (Saccharomyces cerevisiae) branchpoint sequence (5'-UACUAACGUAGUA with the BPS italicized and the branchsite adenosine underlined) base paired with its complementary sequence from U2 snRNA. The structure shows a nearly ideal A-form helix from which two unpaired adenosines flip out. Although the adenosine adjacent to the branchsite adenosine is the one bulged out in the structure described here, either of these adenosines can serve as the nucleophile in mammalian but not in yeast pre-mRNA splicing. In addition, the packing of the bulged RNA helices within the crystal reveals a novel RNA tertiary interaction in which three RNA helices interact through bulged adenosines in the absence of any divalent metal ions.     

The secondary structure of guide RNA molecules from Trypanosoma brucei.

RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is characterized by the insertion and deletion of uridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their primary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially potential RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of four different guide RNAs from Trypanosoma brucei at physiological conditions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molecules despite their different primary sequences, fold into similar structures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions with Tms between 33 and 39 degrees C and transition enthalpies of -32 to -38 kcal/mol. Both terminal ends of the RNAs are single-stranded with the 3' ends possibly adopting a single-stranded, helical conformation. Thus, it appears that the gRNA structures are fine tuned to minimize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permit the assembly with other mitochondrial components into the editing machinery.     

The global structures of a wild-type and poorly functional plant luteoviral mRNA pseudoknot are essentially identical

The helical junction region of a -1 frameshift stimulating hairpin-type mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) is characterized by a novel C27.(G7-C14) loop 2-stem 1 minor groove base triple, which is stacked on a C8+.(G12-C28) loop 1-stem 2 major groove base triple. Substitution of C27 with adenosine reduces frameshifting efficiency to a level just twofold above the slip-site alone. Here, we show that the global structure of the C27A ScYLV RNA is nearly indistinguishable from the wild-type counterpart, despite the fact that the helical junction region is altered and incorporates the anticipated isostructural A27.(G7-C14) minor groove base triple. This interaction mediates a 2.3-A displacement of C8+ driven by an A27 N6-C8+ O2 hydrogen bond as part of an A(n-1).C+.G-Cn base quadruple. The helical junction regions of the C27A ScYLV and the beet western yellows virus (BWYV) pseudoknots are essentially superimposable, the latter of which contains an analogous A25.(G7-C14) minor groove base triple. These results reveal that the global ground-state structure is not strongly correlated with frameshift stimulation and point to a reduced thermodynamic stability and/or enhanced kinetic lability that derives from an altered helical junction architecture in the C27A ScYLV RNA as a significant determinant for setting frameshifting efficiencies in plant luteoviral mRNA pseudoknots.     

The interaction networks of structured RNAs

All pairwise interactions occurring between bases which could be detected in three-dimensional structures of crystallized RNA molecules are annotated on new planar diagrams. The diagrams attempt to map the underlying complex networks of base–base interactions and, especially, they aim at conveying key relationships between helical domains: co-axial stacking, bending and all Watson–Crick as well as non-Watson–Crick base pairs. Although such wiring diagrams cannot replace full stereographic images for correct spatial understanding and representation, they reveal structural similarities as well as the conserved patterns and distances between motifs which are present within the interaction networks of folded RNAs of similar or unrelated functions. Finally, the diagrams could help devising methods for meaningfully transforming RNA structures into graphs amenable to network analysis.