Diversity of base-pair conformations and their occurrence in rRNA structure and RNA structural motifs

In addition to the canonical base-pairs comprising the standard Watson-Crick (C:G and U:A) and wobble U:G conformations, an analysis of the base-pair types and conformations in the rRNAs in the high-resolution crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits has identified a wide variety of non-canonical base-pair types and conformations. However, the existing nomenclatures do not describe all of the observed non-canonical conformations or describe them with some ambiguity. Thus, a standardized system is required to classify all of these non-canonical conformations appropriately. Here, we propose a new, simple and systematic nomenclature that unambiguously classifies base-pair conformations occurring in base-pairs, base-triples and base-quadruples that are associated with secondary and tertiary interactions. This system is based on the topological arrangement of the two bases and glycosidic bonds in a given base-pair. Base-pairs in the internal positions of regular secondary structure helices usually form with canonical base-pair groups (C:G, U:A, and U:G) and canonical conformations (C:G WC, U:A WC, and U:G Wb). In contrast, non-helical base-pairs outside of regular structure helices usually have non-canonical base-pair groups and conformations. In addition, many non-helical base-pairs are involved in RNA motifs that form a defined set of non-canonical conformations. Thus, each rare non-canonical conformation may be functionally and structurally important. Finally, the topology-based isostericity of base-pair conformations can rationalize base-pair exchanges in the evolution of RNA molecules.     

Inosine.adenine base pairs in a B-DNA duplex.

The structure of the synthetic deoxydodecamer d(C-G-C-I-A-A-T-T-A-G-C-G) has been determined by single crystal X-ray diffraction techniques at 2.5A resolution. The refinement converged with a crystallographic residual, R = 0.19 and the location of 64 solvent molecules. The sequence crystallises as a B-DNA helix with 10 Watson-Crick base-pairs (4 A.T. and 6 G.C) and 2 inosine.adenine (I.A) pairs. The present work shows that in the purine.purine base-pairs the adenine adopts syn orientation with respect to the furanose moiety while the inosine is in the trans (anti) orientation. Two hydrogen bonds link the I.A. base-pair, one between N-1(I) and N-7(A), the other between O-6(I) and N-6(A). This bulky purine.purine base-pair is incorporated in the double helix at two positions with little distortion of either local or global conformation. The pairing observed in this study is presented as a model for I.A base-pairs in RNA codon-anticodon interactions and may help explain the thermodynamic stability of inosine containing base-pairs. Conformational parameters and base stacking interactions are presented and where appropriate compared with those of the native compound, d(C-G-C-G-A-A-T-T-C-G-C-G) and with other studies of oligonucleotides containing purine.purine base-pairs.     

Crystal structure and stability of a DNA duplex containing A(anti).G(syn) base-pairs

The synthetic dodecanucleotide d(CGCAAATTGGCG) has been analysed by single-crystal X-ray diffraction techniques and the structure refined to R = 0.16 and 2.25 A resolution, with the location of 94 solvent molecules. The sequence crystallizes as a full turn of a B-DNA helix with ten Watson-Crick base-pairs and two adenine-guanine mispairs. The analysis clearly shows that the mismatches are of the form A(anti).G(syn). Thermal denaturation studies indicate that the stability of the duplex is strongly pH dependent, with a maximum at pH 5.0, suggesting that the base-pair is stabilized by protonation. Three different arrangements have been observed for base-pairs between guanine and adenine and it is likely that A.G mismatch conformation is strongly influenced by dipole-dipole interactions with adjacent base-pairs.     

Alternation of DNA and solvent layers in the A form of d(GGCGCC) obtained by ethanol crystallization

We have determined by X-ray crystallography the structure of the hexamer duplex d(GGCGCC)2 in the A-form using ethanol as a precipitant. The same sequence had previously been crystallized in the B-form, but with 2-methyl-2,4-pentanediol as a precipitant. It appears that ethanol precipitation is a useful method to induce the formation of A-form crystals of DNA. Packing of the molecules in the crystal has unique features: the known interaction of A-DNA duplexes between terminal base-pairs and the minor groove of neighbor molecules is combined with a superstructure consisting in an alternation of DNA layers and solvent layers (water/ions). This organization in layers has been observed before, also with hexamers in the A conformation which crystallize in the same space group (C2221). The solvent layer has a precise thickness, although very few ordered water molecules can be detected. Another feature of this crystal is its large unit cell, which gives rise to an asymmetric unit with three hexamer duplexes. One of the three duplexes is quite different from the other two in several aspects: the number of base pairs per turn, the twist pattern, the mean value of the twist angle and the fact that one terminal base-pair is not stacked as part of the duplex and appears to be disordered. So the variability in conformation of this sequence is remarkable.     

Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton

Using nuclear magnetic resonance line broadening, longitudinal relaxation and magnetization transfer from water, we have measured the imino proton exchange times in the duplex form of the 10-mer d-CGCGATCGCG and in seven other deoxy-duplexes, as a function of the concentration of exchange catalysts, principally ammonia. All exchange times are catalyst dependent. Base-pair lifetimes are obtained by extrapolation to infinite concentration of ammonia. Lifetimes of internal base-pairs are in the range of milliseconds at 35 degrees C and ten times more at 0 degrees C. Lifetimes of neighboring pairs are different, hence base-pairs open one at a time. Lifetimes of d(G.C) are about three times longer than those of d(A.T). The nature of neighbors usually has little effect, but lifetime anomalies that may be related to sequence and/or structure have been observed. In contrast, there is no anomaly in the A.T base-pair lifetimes of d-CGCGA[TA]5TCGCG, a model duplex of poly[d(A-T)].poly[d(A-T)]. The d(A.T) lifetimes are comparable to those of r(A.U) that we reported previously. End effects on base-pair lifetimes are limited to two base-pairs. The low efficiency of exchange catalysts is ascribed to the small dissociation constant of the deoxy base-pairs, and helps to explain why exchange catalysis had been overlooked in the past. This resulted in a hundredfold overestimation of base-pair lifetimes. Cytosine amino proteins have been studied in the duplex of d-CGm5CGCG. Exchange from the closed base-pair is indicated. Hence, the use of an amino exchange rate to evaluate the base-pair dissociation constant would result in erroneous, overestimated values. Catalyzed imino proton exchange is at this time the safest and most powerful, if not the only probe of base-pair kinetics. We propose that the single base-pair opening event characterized here may be the only mode of base-pair disruption, at temperatures well below the melting transition.     

Alternation of DNA and Solvent Layers in the A Form of d(GGCGCC) Obtainhted by Ethanol Crystallization

Abstract

We have determined by X-ray crystallography the structure of the hexamer duplex d(GGCGCC)₂ in the A-form using ethanol as a precipitant. The same sequence had previously been crystallized in the B-form, but with 2-methyl-2, 4-pentanediol as a precipitant. It appears that ethanol precipitation is a useful method to induce the formation of A-form crystals of DNA. Packing of the molecules in the crystal has unique features: the known interaction of A-DNA duplexes between terminal base-pairs and the minor groove of neighbor molecules is combined with a superstructure consisting in an alternation of DNA layers and solvent layers (water/ions). This organization in layers has been observed before, also with hexamers in the A conformation which crystallize in the same space group (C222 ₁). The solvent layer has a precise thickness, although very few ordered water molecules can be detected. Another feature of this crystal is its large unit cell, which gives rise to an asymmetric unit with three hexamer duplexes. One of the three duplexes is quite different from the other two in several aspects: the number of base pairs per turn, the twist pattern, the mean value of the twist angle and the fact that one terminal base-pair is not stacked as part of the duplex and appears to be disordered. So the variability in conformation of this sequence is remarkable.     

The structure of the HIV-1 RRE high affinity rev binding site at 1.6 A resolution

The crystal structure of a 28 nt RNA fragment containing the human immunodeficiency virus type 1 (HIV-1) Rev response element high affinity binding site for Rev protein has been solved at 1.6 A resolution. The overall structure of the RRE helix is greatly distorted from A-form geometry by the presence of two purine-purine base-pairs and two single nucleotide bulges. G48 and G71 form a Hoogsteen-type asymmetric base-pair with G71 adopting a syn conformation. The non-canonical regions in the unliganded Rev response element molecule narrow the major groove width with respect to standard A-RNA. The Rev response element structure observed here represents a closed form of the Rev binding site and differs from conformations of the RNA observed previously by solution NMR studies.     

The conformational variability of an adenosine.inosine base-pair in a synthetic DNA dodecamer.

A crystal structure analysis of the synthetic deoxydodecamer d(CGCAAATTIGCG) which contains two adenosine.inosine (A.I) mispairs has revealed that, in this sequence, the A.I base-pairs adopt a A(anti).I(syn) configuration. The refinement converged at R = 0.158 for 2004 reflections with F greater than or equal to 2 sigma(F) in the range 7.0-2.5A for a model consisting of the DNA duplex and 71 water molecules. A notable feature of the structure is the presence of an almost complete spine of hydration spanning the minor groove of the whole of the (AAATTI)2 core region of the duplex. pH-dependent ultraviolet melting studies have suggested that the base-pair observed in the crystal structure is, in fact, a protonated AH+ (anti).I(syn) species and that the A.I base-pairs in the sequence studied display the same conformational variability as A.G mispairs in the sequence d(CGCAAATTGGCG). The AH+(anti).I(syn) base-pair predominates below pH 6.5 and an A(anti).I(anti) mispair is the major species present between pH 6.5 and 8.0. The protonated base-pairs are held together by two hydrogen bonds one between N6(A) and O6(I) and the other between N1(A) and N7(I). This second hydrogen bond is a direct result of the protonation of the N1 of adenosine. The ultraviolet melting studies indicate that the A(anti).I(anti) base-pair is more stable than the A(anti).G(anti) base-pair but that the AH+(anti).I(syn) base pair is less stable than its AH+(anti).G(syn) analogue. Possible reasons for this observation are discussed.     

A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat

All RNA sequences that fold into hairpins possess the intrinsic potential to form intermolecular duplexes because of their high self-complementarity. The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations. We developed and applied a novel approach to unambiguously distinguish RNA hairpin and duplex conformations for the structural analysis of a Xist RNA A-repeat. Using a combination of a quantitative HNN-COSY experiment and an optimized double isotope-filtered NOESY experiment we could define the conformation of the 26-mer A-repeat RNA. In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat. The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter- and intra-molecular base-pairing patterns.     

The phage 434 Cro/OR1 complex at 2.5 A resolution

The crystal structure of phage 434 Cro protein in complex with a 20 base-pair DNA fragment has been determined to 2.5 A resolution. The DNA fragment contains the sequence of the OR1 operator site. The structure shows a bent conformation for the DNA, straighter at the center and more bent at the ends. The central base-pairs adopt conformations with significant deviations from coplanarity. The two molecules interact extensively along their common interface, both through hydrogen bonds and van der Waals interactions. The significance of these interactions for operator binding and recognition is discussed.     

The influence of helix morphology on co-operative polyamide backbone conformational flexibility in peptide nucleic acid complexes.

A systematic analysis of peptide nucleic acid (PNA) complexes deposited in the Protein Data Bank has been carried out using a set of contiguous atom torsion angle definitions. The analysis is complemented by molecular mechanics adiabatic potential energy calculations on hybrid PNA-nucleic acid model systems. Hitherto unobserved correlations in the values of the (alpha and epsilon) dihedral angles flanking the backbone secondary amide bond are found. This dihedral coupling forms the basis of a PNA backbone conformation classification scheme. Six conformations are thus characterised in experimental structures. Helix morphology is found to exert a significant influence on backbone conformation and flexibility: Watson-Crick PNA strands in complexes with DNA and RNA, that possess A-like base-pair stacking, adopt backbone conformations distinct from those in PNA.DNA-PNA triplex and PNA-PNA duplex P-helix forms. Solvation effects on Watson-Crick PNA backbone conformation in heterotriplexes are discussed and the possible involvement of inter-conformational transitions and dihedral angle uncoupling in asymmetric heteroduplex base-pair breathing is suggested.     

Crystal structure of an RNA duplex r(gugucgcac)(2) with uridine bulges.

The crystal structure of a nonamer RNA duplex with a uridine bulge in each strand, r(gugucgcac)(2), was determined at 1.4 A resolution. The structure was solved by multiple anomalous diffraction phasing method using a three-wavelength data set collected at the Advanced Protein Source and refined to a final R(work)/R(free) of 21.2 %/23.4 % with 33,271 independent reflections (Friedel pairs unmerged). The RNA duplex crystallized in the tetragonal space group P4(1)22 with two independent molecules in the asymmetric unit. The unit cell dimensions are a=b=47.18 A and c=80.04 A. The helical region of the nonamer adopts the A-form conformation. The uridine bulges assume similar conformations, with uracils flipping out and protruding into the minor groove. The presence of the bulge induces very large twist angles (approximately +50 degrees) between the base-pairs flanking the bulges while causing profound kinks in the helix axis at the bulges. This severe twist and the large kink in turn produces a very narrow major groove at the middle of the molecule. The ribose sugars of the guanosines before the bulges adopt the C2'-endo conformation while the rest, including the bulges, are in the C3'-endo conformation. The intrastrand phosphate-phosphate (P-P) distance of the phosphate groups flanking the bulges (approximately 4.4 A) are significantly shorter than the average P-P distance in the duplex (6.0 A). This short distance between the two phosphate groups brings the non-bridging oxygen atoms close to each other where a calcium ion is bound to each strand. The calcium ions in molecule 1 are well defined while the calcium ions in molecule 2 are disordered.     

A 500 MHz proton NMR study of stacking interactions: binding of tripeptide Lys-Tyr-Lys to tetradeoxynucleotide d-GpCpGpC.

The complete sequential assignment and conformation of d-GpCpGpC in D2O has been determined from 1D NMR spectra at 285-320 K and room temperature 2D-COSY and NOESY spectra. The tetradeoxynucleotide exists primarily as a right handed double helix at 285 K, having Tm as 314 K. On binding to a tripeptide Lys-Tyr-Lys in a concentration equimolar to tetranucleotide duplex, the Tyr ring protons shift upfield by 0.14 ppm at 285 K. The increase in Tm on binding suggests stabilization of duplex. The existence of intermolecular NOEs between C4 sugar protons and Tyr alpha C and Lys alpha C protons give direct evidence of proximity of Tyr residue to the C4 base of d-GpCpGpC. The conformation of d-GpCpGpC remains unchanged on binding. The observed results are interpreted in terms of preferential stacking of aromatic ring of Tyr residue with proximal base-pair of d-GpCpGpC, stabilized by electrostatic interaction of Lysine side chains with backbone phosphates. This is in contrast to intercalculation of aromatic dyes within base-pairs resulting in a change in sugar conformation at the binding site.     

M-DNA: A complex between divalent metal ions and DNA which behaves as a molecular wire

M-DNA is a complex of DNA with divalent metal ions (Zn(2+), Co(2+), or Ni(2+)) which forms at pH conditions above 8. Upon addition of these metal ions to B-DNA at pH 8.5, the pH decreases such that one proton is released per base-pair per metal ion. Together with previous NMR data, this result demonstrated that the imino proton in each base-pair of the duplex was substituted by a metal ion and that M-DNA might possess unusual conductive properties. Duplexes of 20 base-pairs were constructed with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Upon formation of M-DNA (with Zn(2+)) the fluorescence of the donor was 95 % quenched. Fluorescence lifetime measurements showed the presence of a very fast component in the decay kinetics with tau相似文献   

Three-dimensional structures of bulge-containing DNA fragments.

The three-dimensional structure of a DNA tridecamer d(CGCAGAATTCGCG)2 containing bulged adenine bases was determined by single crystal X-ray diffraction methods, at 120 K, to 2.6 A resolution. The structure is a B-DNA type double helix with a single duplex in the asymmetric unit. One of the bulged adenine bases loops out from the double helix, while the other stacks in to it. This is in contrast to our preliminary finding, which indicated that both adenine bases were looped out. This revised model was confirmed by the use of a covalently bound heavy-atom derivative. The conformation of the looped-out bulge hardly disrupts base stacking interactions of the bases flanking it. This is achieved by the backbone making a "loop-the-loop" curve with the extra adenine flipping over with respect to the other nucleotides in the strand. The looped-out base intercalates into the stacked-in bulge site of a symmetrically related duplex. The looped-out and stacked-in bases form an A.A reversed Hoogsteen base-pair that stacks between the surrounding base-pairs, thus stabilizing both bulges. The double helix is frayed at one end with the two "melted" bases participating in intermolecular interactions. A related structure, of the same tridecamer, after soaking the crystals with proflavin, was determined to 3.2 A resolution. The main features of this B-DNA duplex are basically similar to the native tridecamer but differ in detail especially in the conformation of the bulged-out base. Accommodation of a large perturbation such as that described here with minimal disruption of the double helix shows both the flexibility and resiliency of the DNA molecule.     

Solvent-accessible surfaces of nucleic acids

Static solvent-accessible surface areas were calculated for DNA and RNA double helices of varied conformation, composition and sequence, for the single helix of poly(rC), and for a transfer RNA. The results show that for DNA and RNA double helices, two thirds of the water-accessible surface area become buried on double helix formation; phosphate oxygens retain near maximal exposure while the bases are 80% buried. Transfer RNA exposes slightly less surface per residue than does double-helical RNA, despite the presence of several additional “modified” groups, all of which are exposed significantly.When a probe corresponding to a single water molecule is used, both the total and atom type exposures are very similar for A-DNA and B-DNA, although marked differences appear in the major and minor groove exposures between the two conformations. For a given base-pair, the accessible surface area buried upon double-helical stacking is nearly constant (within 5%) for different sequences of neighboring base-pairs.For probes larger than single water molecules, there exist considerable differences in the total and atom type exposures of A-DNA and B-DNA. Conformational transitions between the A-DNA and B-DNA helical forms can thus be related to differences in the accessible areas for “structured” water, or a secondary hydration shell, rather than to interactions with individual water molecules of the primary hydration shell. The base-composition dependence of DNA helical conformation can be explained in terms of the opposing effects of thymine methyl groups of A · T base-pairs and the amino groups of G · C base-pairs upon the solvent within the grooves.The area calculations show that primarily the major groove of B-DNA and the minor groove of A-DNA have sufficient accessible surface area to be recognized by a probe size corresponding to the side-chains of amino acids.     

The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges

BACKGROUND: An important step in retroviral replication is dimerization of the genomic RNA prior to encapsidation. Dimerization is initiated by the formation of a transient 'kissing-loop complex' that is thought to be subsequently matured into an extended duplex by the nucleocapsid protein (NCp). Although chemical probing and nuclear magnetic resonance spectroscopy have provided insight into the structure of the kissing-loop structure, no structural information concerning the extended-duplex state is available so far. RESULTS: The structure of a minimal HIV-1 RNA dimerization initiation site has been solved at 2.3 A resolution in two different space groups. It reveals a 22 base pair extended duplex with two noncanonical Watson-Crick-like G-A mismatches, each adjacent to a bulged-out adenine. The structure shows significant asymmetry in deep groove width and G-A base-pair conformations. A network of eight magnesium cations was clearly identified, one being unusually chelated by the 3' phosphate of each bulge across an extremely narrowed deep major groove. CONCLUSIONS: These crystal structures represent the putative matured form of the initial kissing-loop complex. They show the ability of this self-complementary RNA hairpin loop to acquire a more stable extended duplex structure. Both bulged adenines form a striking 'base grip' that could be a recognition signal, either in cis for another viral RNA sequence, or in trans for a protein, possibly the NCp. Magnesium binding might be important to promote and stabilize the observed extrahelical conformation of these bulges.     

Influence of the 2'-hydroxyl group conformation on the stability of A-form helices in RNA

The 2'-hydroxyl group plays fundamental roles in both the structure and the function of RNA, and is the major determinant of the conformational and thermodynamic differences between RNA and DNA. Here, we report a conformational analysis of 2'-OH groups of the HIV-2 TAR RNA by means of NMR scalar coupling measurements in solution. Our analysis supports the existence of a network of water molecules spanning the minor groove of an RNA A-form helix, as has been suggested on the basis of a high-resolution X-ray study of an RNA duplex. The 2'-OH protons of the lower stem nucleotides of the TAR RNA project either towards the O3' or towards the base, where the 2'-OH group can favorably participate in H-bonding interactions with a water molecule situated in the nucleotide base plane. We observe that the k(ex) rate of the 2'-OH proton with the bulk solvent anti-correlates with the base-pair stability, confirming the involvement of the 2'-OH group in a collective network of H-bonds, which requires the presence of canonical helical secondary structure. The methodology and conformational analysis presented here are broadly applicable and facilitate future studies aimed to correlate the conformation of the 2'-OH group with both the structure and the function of RNA and RNA-ligand complexes.     

Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins.

The HIV-1 regulatory proteins tat and rev are both RNA binding proteins which recognize sequences in duplex RNA which are close to structural distortions. Here we identify phosphate contacts which are critical for each binding reaction by use of a new method. Model RNA binding sites are constructed carrying substitutions of individual phosphodiesters by uncharged methylphosphonate derivatives isolated separately as Rp and Sp diastereoisomers and tested for protein binding by competition assays. In the binding of tat to the trans-activation response region (TAR), three phosphates, P21 and P22 which are adjacent to the U-rich bulge and P40 on the opposite strand, are essential and in each case both isomers inhibit binding. Similarly, in the interaction between the HIV-1 rev protein and the rev-responsive element (RRE) both methylphosphonate isomers at P103, P104, P124 and P125 interfere with rev binding. At P106, only the Rp methylphosphonate isomer is impaired in rev binding ability and it is proposed that the Rp oxygen is hydrogen-bonded to an uncharged amino acid or to a main chain hydrogen atom. Synthetic chemistry techniques also provide evidence for the conformations of non-Watson-Crick G106:G129 and G105:A131 base-pairs in the RRE 'bubble' structure upon rev binding. Almost all functional groups on the 5 bulged residues in the bubble have been ruled out as sites of contact with rev but, by contrast, the N7-positions of each G residue in the flanking base-pairs are identified as sites of likely hydrogen-bonding to rev. The results show that both tat and rev recognize the major groove of distorted RNA helixes and that both proteins make specific contacts with phosphates which are displaced from the sites of base-pair contact.