A sulfate pocket formed by three GoU pairs in the 0.97 A resolution X-ray structure of a nonameric RNA

The crystal structure of the RNA duplex [r(CGUGAUCG)dC]2 has been solved at a resolution of 0.97 A. The model has been refined to R-work and R-free of 14.88% and 19.54% for 23,838 independent reflections. The base-pairing scheme forces the 5'-rC to be excluded from the helix and to be disordered. In the crystals, the sequence promotes the formation of two GoU wobble pairs that cluster around a crystallographic threefold axis in two different ways. In the first contact type, the GoU pairs are exclusively surrounded by water molecules, whereas in the other contact type, the three amino groups of the guanine residues of the symmetry-related GoU pairs trap a sulfate ion. This work provides the first example of the interaction of a GoU pair with a sulfate ion in a helical context. Despite the negative charge on the polynucleotide backbone, the guanine amino N2 is able to attract negatively charged groups that could, in the folding of complex RNA molecules, belong to a negative phosphodiester group from a neighboring strand and, in a RNA-protein complex, to a negative carboxyl group of an aspartate or glutamate side chain.     

The multiple flavors of GoU pairs in RNA

Wobble GU pairs (or GoU) occur frequently within double‐stranded RNA helices interspersed within the standard G═C and A─U Watson‐Crick pairs. However, other types of GoU pairs interacting on their Watson‐Crick edges have been observed. The structural and functional roles of such alternative GoU pairs are surprisingly diverse and reflect the various pairings G and U can form by exploiting all the subtleties of their electronic configurations. Here, the structural characteristics of the GoU pairs are updated following the recent crystallographic structures of functional ribosomal complexes and the development in our understanding of ribosomal translation.     

Eukaryotic tRNA sequences present conserved and amino acid-specific structural signatures

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson–Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.     

Wobble base-pairing in codon-anticodon interactions: a theoretical modelling study

The Crick wobble hypothesis attributes the phenomenon of codon degeneracy to a certain impreciseness of pairing between the third base of the codon and the first base of the anticodon. This theoretical study investigates the pairing properties of some wobble bases, including both, observed and unobserved pairs. Some wobble base-pairs are predicted to follow the Watson-Crick pairs in configuration and pairing facility, while others deviate from this norm. The observed U:V pair is unique in that a pairing configuration may be suggested for it wherein the hydrogen-bonding involves the exocyclic 5-carboxymethoxy group of V. By comparing the theoretical data on the configurations of these pairs with the evidence for their existence/non-existence in nature, some guidelines emerge for differentiating between observed and unobserved base pairs on the basis of the pairing configuration.     

Thermodynamic analysis of 5' and 3' single- and 3' double-nucleotide overhangs neighboring wobble terminal base pairs

Thermodynamic parameters are reported for duplex formation of 40 self-complementary RNA duplexes containing wobble terminal base pairs with all possible 3′ single and double-nucleotide overhangs, mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest neighbor analysis, the addition of a single 3′ dangling nucleotide increases the stability of duplex formation up to 1 kcal/mol in a sequence-dependent manner. The addition of a second dangling nucleotide increases the stability of duplexes closed with wobble base pairs in an idiosyncratic manner. The results allow for the development of a nearest neighbor model, which improves the predication of free energy and melting temperature for duplexes closed by wobble base pairs with 3′ single or double-nucleotide overhangs. Phylogenetic analysis of naturally occurring miRNAs was performed. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent on the orientation of the GU closing base pair rather than the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for the 5′ single terminal overhangs adjacent to wobble closing base pairs are also presented.     

Classification of the possible pairs between the first anticodon and the third codon positions based on a simple model assuming two geometries with which the pairing effectively potentiates the decoding complex

Crick's wobble theory states that some specific pairs between the bases at the first position of the anticodon (position 34) and the third position of the codon (position III) are allowed and the others are disallowed during the correct codon recognition. However, later researches have shown that the pairing rule, or the wobble rule, is different from the supposed one. Despite the continuing efforts including computer-aided model building studies and analyses of three-dimensional structures in the crystals of the ribosomes, the structural backgrounds of the wobble rule are still unclear. Here, I classify the possible pairs into 6 classes according to the increases accompanying the formation of the pairs in the potential productivity of the decoding complex on the basis of a simple model that was originally proposed previously and is refined here. In the model, the conformation with the base at position 34 displaced toward the minor groove side from the position for the Watson-Crick pairs is supposed to be equivalent to the conformation with the Watson-Crick pairs. It is also reasoned and supposed that some weak pairs may sometimes be allowed depending on the structural context. It is demonstrated that most of the experimental results reported so far are consistent with the model. I discuss on which experimental facts can be reasoned with the model and which need further explanations. I expect that the model will be a good basis for further understanding of the wobble rule and its structural backgrounds.     

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.     

Crystal structure of a 14 bp RNA duplex with non-symmetrical tandem GxU wobble base pairs

Adjacent GxU wobble base pairs are frequently found in rRNA. Atomic structures of small RNA motifs help to provide a better understanding of the effects of various tandem mismatches on duplex structure and stability, thereby providing better rules for RNA structure prediction and validation. The crystal structure of an RNA duplex containing the sequence r(GGUAUUGC-GGUACC)2 has been solved at 2.1 A resolution using experimental phases. Novel refinement strategies were needed for building the correct solvent model. At present, this is the only short RNA duplex structure containing 5'-U-U-3'/3'-G-G-5' non-symmetric tandem GxU wobble base pairs. In the 14mer duplex, the six central base pairs are all displaced away from the helix axis, yielding significant changes in local backbone conformation, helix parameters and charge distribution that may provide specific recognition sites for biologically relevant ligand binding. The greatest deviations from A-form helix occur where the guanine of a wobble base pair stacks over a purine from the opposite strand. In this vicinity, the intra-strand phosphate distances increase significantly, and the major groove width increases up to 3 A. Structural comparisons with other short duplexes containing symmetrical tandem GxU or GxT wobble base pairs show that nearest-neighbor sequence dependencies govern helical twist and the occurrence of cross-strand purine stacks.     

The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems

The G x U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodynamic stability to Watson-Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G x C or A x U base pairs. The G x U wobble base pair also has unique chemical, structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson-Crick base pairs or other mispairs. These features mark sites containing G x U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biological processes.     

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs. By comparing this structure with the analogous high resolution structure of d(CGCGCG) we can visualize the structural changes as well as the reorganization of the solvent molecules associated with wobble base pairing. There is only a small distortion of the Z-DNA backbone resulting from introduction of the GT mismatched base pairs. The water molecules cluster around the wobble base pair taking up all of the hydrogen bonding capabilities of the bases due to wobble pairing. These bridging water molecules serve to stabilize the base-base interaction and, thus, may be generally important for base mispairing either in DNA or in RNA molecules.     

Theoretical study of the geometrical arrangement of GT and GA wobble pairs in two short duplexes, Proton NMR chemical shifts and interaction energy calculations

NMR shielding constants are calculated for the base protons of duplexes formed by the dodecamer d(CGTGAATTCGCG) and the decamer d(CCAAGATTGG). A good agreement with experimental data is obtained for B-DNA helices in which the wobble GT and GA pairs are in the plane of the corresponding GC pairs of the parent duplexes formed by d(CGCGAATTCGCG) and d(CCAAGCTTGG), if the glycosyl bonds of T and G or A and G are symmetrical with respect to the dyad axis of the Watson-Crick GC pair. Interaction energy calculations show that this type of geometrical arrangement, which implies a distortion of the ribonphosphate backbone of both strands of the duplexes are more stable than those in which only one strand has its conformation modified by the presence of the wobble pair. For the duplex containing the GA pair, NMR chemical shifts as well as interaction energy computations favour the Watson-Crick hydrogen bonding scheme. The variation of the different contributions (intrastrand, interstrand, pair-pair) to the interaction energy between the bases of the duplexes, with the geometrical arrangement of the wobble pairs, is reported.     

Configuration of wobble base pairs having pyrimidines as anticodon wobble bases: significance for codon degeneracy

Degeneracy of the genetic code was attributed by Crick to imprecise hydrogen-bonded base-pairing at the wobble position during codon–anticodon pairing. The Crick wobble rules define but do not explain the RNA base pair combinations allowed at this position. We select six pyrimidine bases functioning as anticodon wobble bases (AWBs) to study their H-bonded pairing properties with the four major RNA bases using density functional theory at the B3LYP/6-31G(d,p) level. This is done to assess the extent to which the configuration of a solitary RNA wobble base pair may in itself determine specificity and degeneracy of the genetic code by allowing or disallowing the given base pair during codon–anticodon pairing. Calculated values of select configuration markers for the base pairs screen well between allowed and disallowed base pairs for most cases examined here, where the base pair width emerges as an important factor. A few allowed wobble pairs invoke the involvement of RNA nucleoside conformation, as well as involvement of the exocyclic substituent in H-bonding. This study, however, cannot explain the disallowed status of the Ura?Gua wobble pair on the basis of configuration alone. Explanation of the allowed status of the V?Ura pair requires further study on the mediatory role of water molecules. Apart from these two cases, these computational results are sufficient, on the basis of base pair configuration alone, to account for the specificity and degeneracy of the genetic code for all known cases of codon–anticodon pairing which involve the pyrimidine AWBs studied here.     

Effects of 5-fluorouracil/guanine wobble base pairs in Z-DNA: molecular and crystal structure of d(CGCGFG).

The chemotherapeutic agent 5-fluorouracil is a DNA base analogue which is known to incorporate into DNA in vivo. We have solved the structure of the oligonucleotide d(CGCGFG), where F is 5-fluorouracil (5FU). The DNA hexamer crystallizes in the Z-DNA conformation at two pH values with the 5FU forming a wobble base pair with guanine in both crystal forms. No evidence of the enol or ionized form of 5FU is found under either condition. The crystals diffracted X-rays to a resolution of 1.5 A and their structures have been refined to R-factors of 20.0% and 17.2%, respectively, for the pH = 7.0 and pH = 9.0 forms. By comparing this structure to that of d(CGCGCG) and d(CGCGTG), we were able to demonstrate that the backbone conformation of d(CGCGFG) is similar to that of the archetypal Z-DNA. The two F-G wobble base pairs in the duplex are structurally similar to the T-G base pairs both with respect to the DNA helix itself and its interactions with solvent molecules. In both cases water molecules associated with the wobble base pairs bridge between the bases and stabilize the structure. The fluorine in the 5FU base is hydrophobic and is not hydrogen bonded to any solvent molecules.     

The electrostatic characteristics of G.U wobble base pairs

G·U wobble base pairs are the most common and highly conserved non-Watson–Crick base pairs in RNA. Previous surface maps imply uniformly negative electrostatic potential at the major groove of G·U wobble base pairs embedded in RNA helices, suitable for entrapment of cationic ligands. In this work, we have used a Poisson–Boltzmann approach to gain a more detailed and accurate characterization of the electrostatic profile. We found that the major groove edge of an isolated G·U wobble displays distinctly enhanced negativity compared with standard GC or AU base pairs; however, in the context of different helical motifs, the electrostatic pattern varies. G·U wobbles with distinct widening have similar major groove electrostatic potentials to their canonical counterparts, whereas those with minimal widening exhibit significantly enhanced electronegativity, ranging from 0.8 to 2.5kT/e, depending upon structural features. We propose that the negativity at the major groove of G·U wobble base pairs is determined by the combined effect of the base atoms and the sugar-phosphate backbone, which is impacted by stacking pattern and groove width as a result of base sequence. These findings are significant in that they provide predictive power with respect to which G·U sites in RNA are most likely to bind cationic ligands.     

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.     

Crystal structure of an RNA duplex r(G GCGC CC)2 with non-adjacent G*U base pairs

The crystal structure of a self-complementary RNA duplex r(GGGCGCUCC)2with non-adjacent G*U and U*G wobble pairs separated by four Watson-Crick base pairs has been determined to 2.5 A resolution. Crystals belong to the space group R3; a = 33.09 A,alpha = 87.30 degrees with a pseudodyad related duplex in the asymmetric unit. The structure was refined to a final Rworkof 17.5% and Rfreeof 24.0%. The duplexes stack head-to-tail forming infinite columns with virtually no twist at the junction steps. The 3'-terminal cytosine nucleosides are disordered and there are no electron densities, but the 3' penultimate phosphates are observed. As expected, the wobble pairs are displaced with guanine towards the minor groove and uracil towards the major groove. The largest twist angles (37.70 and 40.57 degrees ) are at steps G1*C17/G2*U16 and U7*G11/C8*G10, while the smallest twist angles (28.24 and 27.27 degrees ) are at G2*U16/G3*C15 and C6*G12/U7*G11 and conform to the pseudo-dyad symmetry of the duplex. The molecule has two unequal kinks (17 and 11 degrees ) at the wobble sites and a third kink at the central G5 site which may be attributed to trans alpha (O5'-P), trans gamma (C4'-C5') backbone conformations. The 2'-hydroxyl groups in the minor groove form inter-column hydrogen bonding, either directly or through water molecules.     

Thermodynamics and kinetics for base-pair opening in the P1 duplex of the Tetrahymena group I ribozyme

The thermodynamics and kinetics for base-pair opening of the P1 duplex of the Tetrahymena group I ribozyme were studied by NMR hydrogen exchange experiments. The apparent equilibrium constants for base pair opening were measured for most of the imino protons in the P1 duplex using the base catalysts NH3, HPO4(2-) or TRIS. These equilibrium constants were also measured for several modified P1 duplexes, and the C-2.G23 base pair was the most stable base pair in all the duplexes. The conserved U-1*G22 base pair is required for activity of the ribozyme and the data here show that this wobble base pair destabilizes neighboring base pairs on only one side of the wobble. A 2'-OMe modification on the U-3 residue stabilized its own base pair but had little effect on the neighboring base pairs. Three base pairs, U-1*G22, C-2*G23 and A2*U21 showed unusual equilibrium constants for opening and possible implications of the opening thermodynamics of these base pairs on the undocking rates of the P1 helix with catalytic core are discussed.