Nuclear magnetic resonance spectroscopy and molecular modeling reveal that different hydrogen bonding patterns are possible for G.U pairs: one hydrogen bond for each G.U pair in r(GGCGUGCC)(2) and two for each G.U pair in r(GAGUGCUC)(2)

G.U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G.U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G.U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G.U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C, the motif 5'-CGUG-3' is less stable by approximately 3 kcal/mol compared with other symmetric tandem G.U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The solution structures of r(GAGUGCUC)(2) and r(GGCGUGCC)(2) duplexes have been determined by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G.U pair region. The striking discovery is that in r(GGCGUGCC)(2) each G.U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addition, the temperature dependence of the imino proton resonances is also consistent with the different number of hydrogen bonds in the G.U pair. To test the NMR models, U or G in various G.U pairs were individually replaced by N3-methyluridine or isoguanosine, respectively, thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.     

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.     

Structural characterisation of the bromouracil.guanine base pair mismatch in a Z-DNA fragment.

The deoxyoligonucleotide d(BrU-G-C-G-C-G) was crystallised at pH 8.2 and its structure analysed by X-ray diffraction. The unit cell, of dimensions a = 17.94, b = 30.85, c = 49.94A contains four DNA duplexes in space group P2(1)2(1)2(1). The duplexes are in the Z conformation, with four Watson-Crick G.C base pairs and two BrU.G base pairs. The structure was refined to an R factor of 0.16 at a resolution of 2.2A with 64 solvent molecules located. The BrU.G base pair mismatch is of the wobble type, with both bases in the major tautomer form and hydrogen bonds linking 0-2 of BrU with N-1 of G and N3 of BrU with 0-6 of G. There is no indication of the presence of ionised base pairs, in spite of the high pH of crystallisation. The results are discussed in terms of the mutagenic properties of 5- bromouracil.     

Stacking of Crick Wobble pair and Watson-Crick pair: stability rules of G-U pairs at ends of helical stems in tRNAs and the relation to codon-anticodon Wobble interaction.

The occurrence of the noncomplementary G-U base pair at the end of a helix is found to be governed by stacking interactions. As a rule, a G-U pair with G on the 5'-side of a Watson-Crick base pair exhibits strikingly greater stacking overlap with the Watson-Crick base pair than a G-U pair on the 3'-side of a Watson-Crick base pair. The former arrangement is expected to be more stable and indeed is observed 29 times out of 32 in the known transfer RNA molecules. In accordance with this rule, the major wobble base pairs G-U or I-U in codon-anticodon interactions have G or I on the 5'-side of the anticodon. Similarly, in initiator tRNAs, this rule is obeyed where now the G is the first letter of the codon (5'-side). In the situation where U is in the wobble position of the anticodon, it is usually substituted at C(5) andmay also have a 2-thio group and it can read one to four codons depending on its modifications. A G at the wobble position of the anticodon can recognize the two codons ending with U or C and modification of G (unless it is I) does not change its reading properties.     

Influence of an exocyclic guanine adduct on the thermal stability, conformation, and melting thermodynamics of a DNA duplex.

As part of an overall program to characterize the impact of mutagenic lesions on the physiochemical properties of DNA, we report here the results of a comparative spectroscopic study on pairs of DNA duplexes both with and without an exocyclic guanine lesion. Specifically, we have studied a family of four 13-mer duplexes of the form d(CGCATGYGTACGC).d(GCGTACZCATGCG) in which Y is either the normal deoxyguanosine residue (G) or the exocyclic guanine adduct 1,N2-propanodeoxyguanosine (X), while Z is either deoxycytosine (C) or deoxyadenosine (A). Thus, the four duplexes studied, which can be designated by the identity of their central Y.Z base pair, are a Watson-Crick duplex (GC), a duplex with a central mismatch (GA), and two duplexes with exocyclic guanine lesions (X), that differ only by the base opposite the lesion (XC and XA). The data derived from our spectroscopic measurements on these four duplexes have allowed us to evaluate the influence of the exocyclic guanine lesion, as well as the base opposite the lesion, on the conformation, thermal stability, and melting energetics of the host DNA duplex. To be specific, our circular dichroism (CD) spectra show that the exocyclic guanine lesion induces alterations in the duplex structure, while our temperature-dependent optical measurements reveal that these lesion-induced structural alterations reduce the thermal stability, the transition enthalpy, and the transition free energy of the duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.C pairing in dodecanucleotide duplexes

The pairing of O6etG with C located four base pairs in from either end of the self-complementary d(C1-G2-C3-O6etG4-A5-G6-C7-T8-C9-G10-C11-G12) duplex (designated O6etG.C 12-mer) has been investigated from an analysis of proton and phosphorus two-dimensional NMR experiments. The structural consequences of increasing the alkyl group size were elucidated from a comparative study of the pairing of O6meG4 with C9 in a related sequence (designated O6meG.C 12-mer). The NMR parameters for both O6alkG-containing dodecanucleotides are also compared with those of the control sequence containing G4.C9 base pairs (designated G.C 12-mer). The NOE cross-peaks detected in the two-dimensional NOESY spectra of the O6alkG.C 12-mer duplexes in H2O solution establish that the O6etG4/O6meG4 and C9 bases at the lesion site stack into the helix between the flanking C3.G10 and A5.T8 Watson-Crick base pairs. The amino protons of C9 at the O6alkG4-C9 lesion site resonate as an average resonance at 7.78 and 7.63 ppm in the O6etG.C 12-mer and O6meG.C 12-mer duplexes, respectively. The observed NOEs between the amino protons of C9 and the CH3 protons of O6alkG4 establish a syn orientation of the O6-alkyl group with respect to the N1 of alkylated guanine. A wobble alignment of the O6alkG4.C9 base pair stablized by two hydrogen bonds, one between the amino group of C9 and N1 of O6alkG and the other between the amino group of O6alkG and N3 of C9, is tentatively proposed on the basis of the NOEs between the amino protons of C9 at the lesion site and the imino protons of flanking Watson-Crick base pairs. The proton and phosphorus chemical shift differences between the O6etG.C 12-mer and O6meG.C 12-mer duplexes are small compared to the differences between these O6alkG-containing duplexes and the control G.C 12-mer duplex.     

To Wobble or Not to Wobble: Modified Bases Incorporated into DNA

Abstract

The ambivalent base analogue P was incorporated in the d(CGCGPG) hexamer to investigate the G.P base pair geometry by X-ray diffraction. Both Watson-Crick and wobble geometries have been found for the crystallographic independent G.P base pairs.     

Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

X-Ray analysis of d(CGCGAATTXGCG)(2) containing a 2' or minute-deoxy-N(4)-methoxycytosine residue at X: a characteristic pattern of sugar puckers in the crystalline state of the Dickerson-Drew type DNA dodecamers

In a series of structural studies on damaged DNA, a modified Dickerson-Drew dodecamer with the sequence d(CGCGAATTmo(4)CGCG), where mo(4)C is 2'-deoxy-N(4)-methoxycytidine, was synthesized and its structure in a new crystal form has been determined by the X-ray diffraction method. The two dodecamers form a B-form duplex, in which the two mo(4)C residues, respectively, form a wobble pair and a Watson-Crick type pair with the guanine residues of the opposite strand. A comparison of the sugar conformations with those of the other related Dickerson-Drew dodecamers indicates a common feature of their puckering patterns. The sugar pucker of the third residue always adopts an intermediate state (C4'-exo-O4'-endo) between the A-form and B-form. This deviation is ascribed to the stacking interaction of the ribose ring at the third residue with the guanine base at the 12th residue, which is brought about by an extra G12:G2 interaction between two duplexes related by a crystallographic 2(1) symmetry.     

NMR structures of r(GCAGGCGUGC)2 and determinants of stability for single guanosine-guanosine base pairs

Nucleotides in RNA that are not Watson-Crick-paired form unique structures for recognition or catalysis, but determinants of these structures and their stabilities are poorly understood. A single noncanonical pair of two guanosines (G) is more stable than other noncanonical pairs and can potentially form pairing structures with two hydrogen bonds in four different ways. Here, the energetics and structure of single GG pairs are investigated in several sequence contexts by optical melting and NMR. The data for r(5'GCAGGCGUGC3')(2), in which G4 and G7 are paired, are consistent with a model in which G4 and G7 alternate syn glycosidic conformations in a two-hydrogen-bond pair. The two distinct structures are derived from nuclear Overhauser effect spectroscopic distance restraints coupled with simulated annealing using the AMBER 95 force field. In each structure, the imino and amino protons of the anti G are hydrogen bonded to the O6 and N7 acceptors of the syn G, respectively. An additional hydrogen-bond connects the syn G amino group to the 5' nonbridging pro-R(p) phosphate oxygen. The GG pair fits well into a Watson-Crick helix. In r(5'GCAGGCGUGC3')(2), the G4(anti), G7(syn) structure is preferred over G4(syn), G7(anti). For single GG pairs in other contexts, exchange processes make interpretation of spectra more difficult but the pairs are also G(syn), G(anti). Thermodynamic data for a variety of duplexes containing pairs of G, inosine, and 7-deazaguanosine flanked by GC pairs are consistent with the structural and energetic interpretations for r(5'GCAGGCGUGC3')(2), suggesting similar GG conformations.     

Database of non-canonical base pairs found in known RNA structures

Atomic resolution RNA structures are being published at an increasing rate. It is common to find a modest number of non-canonical base pairs in these structures in addition to the usual Watson-Crick pairs. This database summarizes the occurrence of these rare base pairs in accordance with standard nomenclature. The database, http://prion.bchs.uh.edu/, contains information such as sequence context, sugar pucker conformation, anti / syn base conformations, chemical shift, p K (a)values, melting temperature and free energy. Of the 29 anticipated pairs with two or more hydrogen bonds, 20 have been encountered to date. In addition, four unexpected pairs with two hydrogen bonds have been reported bringing the total to 24. Single hydrogen bond versions of five of the expected geometries have been encountered among the single hydrogen bond interactions. In addition, 18 different types of base triplets have been encountered, each of which involves three to six hydrogen bonds. The vast majority of the rare base pairs are antiparallel with the bases in the anti configuration relative to the ribose. The most common are the GU wobble, the Sheared GA pair, the Reverse Hoogsteen pair and the GA imino pair.     

NMR studies of triple-strand formation from the homopurine-homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4

The complexes formed by the homopurine and homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4 have been investigated by one- and two-dimensional 1H NMR. Under appropriate conditions [low pH, excess d(TC)4 strand] the oligonucleotides form a triplex containing one d(GA)4 and two d(TC)4 strands. The homopurine and one of the homopyrimidine strands are Watson-Crick base paired, and the second homopyrimidine strand is Hoogsteen base paired in the major groove to the d(GA)4 strand. Hoogsteen base pairing in GC base pairs requires hemiprotonation of C; we report direct observation of the C+ imino proton in these base pairs. Both homopyrimidine strands have C3'-endo sugar conformations, but the purine strand does not. The major triplex formed appears to have four TAT and three CGC+ triplets formed by binding of the second d(TC)4 strand parallel to the d(GA)4 strand with a 3' dangling end. In addition to the triplexes formed, at least one other heterocomplex is observed under some conditions.     

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.     

Multiple structures for the 2-aminopurine-cytosine mispair

The base pair formed between 2-aminopurine (2AP) and cytosine (C) is an intermediate in transition mutations generated by 2AP. To date, several structures have been proposed for the 2AP-C mispair, including those involving a rare tautomer, a protonated base pair, and a neutral wobble structure. In this paper, we describe a series of UV, fluorescence, and NMR studies which demonstrate that an equilibrium exists between the neutral wobble and the protonated Watson-Crick structures. The apparent pK value for the transition between the structures is 5.9-6.0. Formation of a Watson-Crick base pair is accomplished predominantly by protonation of the 2AP residue as indicated by UV spectral changes, fluorescence quenching, and changes in proton chemical shifts. Rapid transfer of the shared proton between the 2AP and cytosine residues is indicated by the rapid exchange of the cytosine amino protons of the protonated Watson-Crick configuration. The relative contribution of the neutral wobble and protonated Watson-Crick configurations to 2AP-induced transition mutations is discussed.     

Probing the role of a secondary structure element at the 5'- and 3'-splice sites in group I intron self-splicing: the tetrahymena L-16 ScaI ribozyme reveals a new role of the G.U pair in self-splicing

Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogues, catalyzes a reaction analogous to the first step of self-splicing, in which a 5'-splice site analogue (S) and guanosine (G) are converted into a 5'-exon analogue (P) and GA. This ribozyme preserves the active site but lacks a short 5'-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5'-splice site in the first step of self-splicing, and P10 forms at the 3'-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms, we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5'- and 3'-splice site analogues that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogues that do not form additional base pairs with the IGS extension. Analysis of a U.U pair immediately 3' to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5'-splice site analogues that form only a subset of base pairs reveal that inclusion of the conserved G.U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G.U step. These results suggest a previously unrecognized role of the G.U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.     

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.     

Thermodynamics of RNA internal loops with a guanosine-guanosine pair adjacent to another noncanonical pair

Thermodynamic parameters measured by optical melting are reported for formation of RNA duplexes containing tandem noncanonical pairs with at least one guanosine-guanosine (GG) pair. For selected sequences, imino proton NMR provides evidence that the desired duplex forms and that the structure of a GG pair adjacent to a noncanonical pair depends on context. A GG pair next to a different noncanonical pair is more stable than expected from measurements of adjacent GG pairs. This is likely due to an unfavorable stacking interaction between adjacent GG pairs, where areas of high negative charge probably overlap. The results suggest a model where tandem noncanonical pairs closed by two GC pairs are assigned the following free energy increments at 37 degrees C: 0.8 kcal/mol for adjacent GG pairs, 1.0 kcal/mol for GG next to UU, and -0.3 kcal/mol for all others. These values are adjusted by 0.65 kcal/mol for each closing AU pair.