Solution structure of dAATAA and dAAUAA DNA bulges

The NMR structure analysis is described for two DNA molecules of identical stem sequences with a five base loop containing a pyrimidine, thymin or uracil, in between purines. These five unpaired nucleotides are bulged out and are known to induce a kink in the duplex structure. The dAATAA bulge DNA is kinked between the third and the fourth nucleotide. This contrasts with the previously studied dAAAAA bulge DNA where we found a kink between the fourth and fifth nucleotide. The total kinking angle is ~104° for the dAATAA bulge. The findings were supported by electrophoretic data and fluorescence resonance energy transfer measurements of a similar DNA molecule end-labeled by suitable fluorescent dyes. For the dAAUAA bulge the NMR data result in a similar structure as reported for the dAATAA bulge with a kinking angle of ~87°. The results are discussed in comparison with a rAAUAA RNA bulge found in a group I intron. Generally, the sequence-dependent structure of bulges is important to understand the role of DNA bulges in protein recognition.     

Non-nearest-neighbor dependence of the stability for RNA bulge loops based on the complete set of group I single-nucleotide bulge loops

Fifty-nine RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters DeltaH degrees, DeltaS degrees, DeltaG 37 degrees, and TM for each sequence were determined. Sequences from this study were combined with sequences from previous studies [Longfellow, C. E., et al. (1990) Biochemistry 29, 278-285; Znosko, B. M., et al. (2002) Biochemistry 41, 10406-10417], thus examining all possible group I single-nucleotide bulge loop and nearest-neighbor sequence combinations. The free energy increments at 37 degrees C for the introduction of a group I single-nucleotide bulge loop range between 1.3 and 5.2 kcal/mol. The combined data were used to develop a model for predicting the free energy of a RNA duplex containing a single-nucleotide bulge. For bulge loops with adjacent Watson-Crick base pairs, neither the identity of the bulge nor the nearest-neighbor base pairs had an effect on the influence of the bulge loop on duplex stability. The proposed model for prediction of the stability of a duplex containing a bulged nucleotide was primarily affected by non-nearest-neighbor interactions. The destabilization of the duplex by the bulge was related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. The stability of a duplex containing a bulged nucleotide adjacent to a wobble base pair also was primarily affected by non-nearest-neighbor interactions. Again, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. However, when one or both of the bulge nearest neighbors was a wobble base pair, the free energy increment for insertion of a bulge loop is dependent upon the position and orientation of the wobble base pair relative the bulged nucleotide. Bulge sequences of the type ((5'UBX)(3'GY)), ((5'GBG)(3'UU)) and ((5'UBU)(3'GG)) are less destabilizing by 0.6 kcal/mol, and bulge sequences of the type ((5'GBX)(3'UY)) and ((5'XBU)(3'YG)) are more destabilizing by 0.4 kcal/mol than bulge loops adjacent to Watson-Crick base pairs.     

An examination of coaxial stacking of helical stems in a pseudoknot motif: the gene 32 messenger RNA pseudoknot of bacteriophage T2

The RNA pseudoknot located at the 5' end of the gene 32 messenger RNA of bacteriophage T2 contains two A-form helical stems connected by two loops, in an H-type pseudoknot topology. A combination of multidimensional NMR methods and isotope labeling were used to investigate the pseudoknot structure, resulting in a more detailed structural model than provided by earlier homonuclear NMR studies. Of particular significance, the interface between the stacked helical stems within the pseudoknot motif is described in detail. The two stems are stacked in a coaxial manner, with an approximately 18 degrees rotation of stem1 relative to stem2 about an axis that is parallel to the helical axis. This rotation serves to relieve what would otherwise be a relatively close phosphate-phosphate contact at the junction of the two stems, while preserving the stabilizing effects of base stacking. The ability of the NMR data to determine pseudoknot bending was critically assessed. The data were found to be a modestly precise indicator of pseudoknot bending, with the angle between the helical axes of stem1 and stem2 being in the range of 15+/-15 degrees. Pseudoknot models with bend angles within this range are equally consistent with the data, since they differ by only small amounts in the relatively short-range interproton distances from which the structure was derived. The gene 32 messenger RNA pseudoknot was compared with other RNA structures with coaxial or near-coaxial stacked helical stems.     

The solution structure of a d[C(TTCG)G] DNA hairpin and comparison to the unusually stable RNA analogue.

The solution structure of the DNA analogue of the unusually stable r[C(UUCG)G] RNA hairpin, 5'-d[GGA-C(TTCG)GTCC]-3', has been determined by NMR spectroscopy, and its structure has been compared to that of the RNA molecule. The RNA molecule is compact and rigid with a highly structured loop. However, the DNA molecule is much less structured. The DNA hairpin contains a B-form stem of four base pairs. The terminal base pair frays, and the 3'-terminal nucleotides, C11 and C12, are in equilibrium between 2'-endo and 3'-endo conformations. Unlike the RNA loop, the DNA loop contains no syn nucleotides, and there is no evidence for base-base or base-phosphate hydrogen bonding in the loop. The loop is flexible, and reveals no specific internucleotide interactions.     

Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting

RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.     

The stereochemistry of a four-way DNA junction: a theoretical study.

The stereochemical conformation of the four-way helical junction in DNA (the Holliday junction; the postulated central intermediate of genetic recombination) has been analysed, using molecular mechanical computer modelling. A version of the AMBER program package was employed, that had been modified to include the influence of counterions and a global optimisation procedure. Starting from an extended planar structure, the conformation was varied in order to minimise the energy, and we discuss three structures obtained by this procedure. One structure is closely related to a square-planar cross, in which there is no stacking interaction between the four double helical stems. This structure is probably closely similar to that observed experimentally in the absence of cations. The remaining two structures are based on related, yet distinct, conformations, in which there is pairwise coaxial stacking of neighbouring stems. In these structures, the four DNA stems adopt the form of two quasi-continuous helices, in which base stacking is very similar to that found in standard B-DNA geometry. The two stacked helices so formed are not aligned parallel to each other, but subtend an angle of approximately 60 degrees. The strands that exchange between one stacked helix and the other are disposed about the smaller angle of the cross (i.e. 60 degrees rather than 120 degrees), generating an approximately antiparallel alignment of DNA sequences. This structure is precisely the stacked X-structure proposed on the basis of experimental data. The calculations indicate distortions from standard B-DNA conformation that are required to adopt the stacked X-structure; a widening of the minor groove at the junction, and reorientation of the central phosphate groups of the exchanging strands. An important feature of the stacked X-structure is that it presents two structurally distinct sides. These may be recognised differently by enzymes, providing a rationalisation for the points of cleavage by Holliday resolvases.     

Solution structure of a consensus stem-loop D RNA domain that plays important roles in regulating translation and replication in enteroviruses and rhinoviruses

Stem-loop D from the cloverleaf RNA is a highly conserved domain within the 5'-UTR of enteroviruses and rhinoviruses. Interaction between the stem-loop D RNA and the viral 3C or 3CD proteins constitutes an essential feature of a ribonucleoprotein complex that plays a critical role in regulating viral translation and replication. Here we report the solution NMR structure of a 38-nucleotide RNA with a sequence that encompasses the entire stem-loop D domain and corresponds to the consensus sequence found in enteroviruses and rhinoviruses. Sequence variants corresponding to Poliovirus type 1 and Coxsackievirus B3 have virtually the same structure, based on small differences in chemical shifts. A substantial number (136) of (1)H-(13)C one-bond residual dipolar coupling (RDC) values were used in the structure determination in addition to conventional distance and torsion angle restraints. Inclusion of the RDC restraints was essential for achieving well-defined structures, both globally and locally. The structure of the consensus stem-loop D is an elongated A-type helical stem capped by a UACG tetraloop with a wobble UG closing base pair. Three consecutive pyrimidine base pairs (two UU and one CU pair) are present in the middle of the helical stem, creating distinctive local structural features such as a dramatically widened major groove. A dinucleotide bulge is located near the base of the stem. The bulge itself is flexible and not as well defined as the other parts of the molecule, but the flanking base pairs are intact. The peculiar spatial arrangement of the distinctive structural elements implies that they may work synergistically to achieve optimal binding affinity and specificity toward the viral 3C or 3CD proteins.     

Thermal stability of RNA hairpins containing a four-membered loop and a bulge nucleotide

Fourteen RNA hairpins containing a four-membered loop and a bulge nucleotide were synthesized and their thermal stabilities determined. The combined contribution of a four-membered loop and bulge A to the free energy of a hairpin is calculated to be 9.3 kcal/mol at 37 degrees C and successfully predicts the stability of an independent RNA hairpin. The introduction of a bulge nucleotide to the helical stem of an RNA hairpin destabilizes the molecule in a sequence-dependent manner. The individual thermodynamic contributions of a four-membered loop and bulge A, G, and U residues to the stability of an RNA hairpin loop are presented.     

Conformational dynamics of a 5S rRNA hairpin domain containing loop D and a single nucleotide bulge

Molecular modeling and molecular dynamics have been employed to study the conformation and flexibility of a 15-nucleotide fragment of the plant 5S rRNA containing loop D and a single uridine bulge. Two different model built initial structures were used: one with the bulge localized inside the helical stem and another with the bulge pointing out from the helix. Several independent 700-ps-long trajectories in aqueous solution with Na(+) conterions were produced for each starting structure. The bulge nucleotide inside the helix stayed in two main conformations, both of which affected the geometry of the stem part opposite the bulge. When the bulge nucleotide was located outside the helix, we found high base mobility and local backbone flexibility. The dynamics of the hydrogen bond network and conformational changes from a direct to a water mediated hydrogen bond in the sheared G-A basepair in the tetraloop was described. Our results correlate with lead ion induced cleavage patterns in 5S rRNA. Sites resistant to nonspecific lead cleavage appeared in all our simulations as the most rigid fragments independent of the localization of the bulge nucleotide.     

Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression

Our previous studies have shown that tandem Alu repeats inhibit green fluorescent protein (GFP) gene expression when inserted downstream of the GFP gene in the pEGFP-C1 vector. We found that the 22R sequence (5'-GTGAAAAAAATGCTTTATTTGT-3') from the antisense PolyA (240 bp polyadenylation signal) of simian virus 40, eliminated repression of GFP gene expression when inserted between the GFP gene and the Alu repeats. The 22R sequence contains an imperfect palindrome; based on RNA structure software prediction, it forms an unstable stem-loop structure, including a loop, a first stem, a bulge, and a second stem. Analysis of mutations of the loop length of the 22R sequence showed that the three-nucleotide loop (wild-type, 22R) induced much stronger GFP expression than did other loop lengths. Two mutations, 4TMI (A7→T, A17→T) and 5AMI (A6→T, T18→A), which caused the base type changes in the bulge and in the second stem in the 22R sequence, induced stronger GFP gene expression than 22R itself. Mutation of the bulge base (A17→T), leading to complete complementation of the stem, caused weaker GFP gene expression. Sequences without a palindrome (7pieA, 5'-GTGAAAAAAATG CAAAAAAAGT-3', 7pieT, 5'-GTGTTTTTTTTGCTTTTTTTGT-3') did not activate GFP gene expression. We conclude that an imperfect palindrome affects and can increase GFP gene expression.     

Solution structure of a trans-opened (10S)-dA adduct of (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a fully complementary DNA duplex: evidence for a major syn conformation

Two-dimensional NMR was used to determine the solution structure of an undecanucleotide duplex, d(CGGTCACGAGG).d(CCTCGTGACCG), in which (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is covalently bonded to the exocyclic N(6)() amino group of the central deoxyadenosine, dA(6), through trans addition at C10 of the epoxide (to give a 10S adduct). The present study represents the first NMR structure of a benzo[a]pyrene (10S)-dA adduct in DNA with a complementary T opposite the modified dA. Exchangeable and nonexchangeable protons of the modified duplex were assigned by the use of TOCSY (in D(2)O) and NOESY spectra (in H(2)O and D(2)O). Sequential NOEs expected for a B-type DNA conformation with typical Watson-Crick base pairing are observed along the duplex, except at the lesion site. We observed a strong intraresidue NOE cross-peak between H1' and H8 of the modified dA(6). The sugar H2' and H2' ' of dC(5) lacked NOE cross-peaks with H8 of dA(6) but showed weak interactions with H2 of dA(6) instead. In addition, the chemical shift of the H8 proton (7.51 ppm) of dA(6) appears at a higher field than that of H2 (8.48 ppm). These NOE and chemical shift data for the dA(6) base protons are typical of a syn glycosidic bond at the modified base. Restrained molecular dynamics/energy minimization calculations show that the hydrocarbon is intercalated from the major groove on the 3'-side of the modified base between base pairs A(6)-T(17) and C(7)-G(16) and confirm the syn glycosidic angle (58 degrees ) of the modified dA(6). In the syn structure, a weak A-T hydrogen bond is possible between the N3-H proton of T(17) and N7 of dA(6) (at a distance of 3.11 A), whereas N1, the usual hydrogen bonding partner for N3-H of T when dA is in the anti conformation, is 6.31 A away from this proton. The 10(S)-dA modified DNA duplex remains in a right-handed helix, which bends in the direction of the aliphatic ring of BaP at about 42 degrees from the helical axis. ROESY experiments provided evidence for interconversion between the major, syn conformer and a minor, possibly anti, conformer.     

Thermal stability, structural features, and B-to-Z transition in DNA tetraloop hairpins as determined by optical spectroscopy in d(CG)(3)T(4)(CG)(3) and d(CG)(3)A(4)(CG)(3) oligodeoxynucleotides

NMR and CD data have previously shown the formation of the T(4) tetraloop hairpin in aqueous solutions, as well as the possibility of the B-to-Z transition in its stem in high salt concentration conditions. It has been shown that the stem B-to-Z transition in T(4) hairpins leads to S (south)- to N (north)-type conformational changes in the loop sugars, as well as anti to syn orientations in the loop bases. In this article, we have compared by means of UV absorption, CD, Raman, and Fourier transform infrared (FTIR), the thermodynamic and structural properties of the T(4) and A(4) tetraloop hairpins formed in 5'-d(CGCGCG-TTTT-CGCGCG)-3' and 5'-d(CGCGCG-AAAA-CGCGCG)-3', respectively. In presence of 5M NaClO(4), a complete B-to-Z transition of the stems is first proved by CD spectra. UV melting profiles are consistent with a higher thermal stability of the T(4) hairpin compared to the A(4) hairpin. Order-to-disorder transition of both hairpins has also been analyzed by means of Raman spectra recorded as a function of temperature. A clear Z-to-B transition of the stem has been confirmed in the T(4) hairpin, and not in the A(4) hairpin. With a right-handed stem, Raman and FTIR spectra have confirmed the C2'-endo/anti conformation for all the T(4) loop nucleosides. With a left-handed stem, a part of the T(4) loop sugars adopt a N-type (C3'-endo) conformation, and the C3'-endo/syn conformation seems to be the preferred one for the dA residues involved in the A(4) tetraloop.     

Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA.     

X-ray and NMR studies of the DNA oligomer d(ATATAT): Hoogsteen base pairing in duplex DNA

We present and analyze the structure of the oligonucleotide d(ATATAT) found in two different forms by X-ray crystallography and in solution by NMR. We find that in both crystal lattices the oligonucleotide forms an antiparallel double helical duplex in which base pairing is of the Hoogsteen type. The double helix is apparently very similar to the standard B-form of DNA, with about 10 base pairs per turn. However, the adenines in the duplex are flipped over; as a result, the physicochemical features of both grooves of the helix are changed. In particular, the minor groove is narrow and hydrophobic. On the other hand, d(ATATAT) displays a propensity to adopt the B conformation in solution. These results confirm the polymorphism of AT-rich sequences in DNA. Furthermore, we show that extrahelical adenines and thymines can be minor groove binders in Hoogsteen DNA.     

S-N transition of the saccharide ring in B-form DNA

A conformational transition of a single deoxyribose was analyzed in B-form trimers dA3:dT3 and dG3:dC3, both in the purine and pyrimidine chains. The main results were obtained for the duplexes with frozen ends, which could be extended by regular double helixes. The geometry of the central sugar ring in the duplexes may strongly deviate from the regular conformation. When deoxyribose changed its conformation in the central pyrimidine, the energy increase was proved to be less significant in comparison with that for purine. In the case of Thy, a decrease in pseudorotation angle P from 140 to 80 degrees causes the energy increase of 0.5 kcal/mol only, the barrier being 1.2 kcal/mol. The energy profile for Cyt has several local minima. The results of calculations were compared with numerous experimental data, they help to explain some NMR data. A perturbation of the duplex AAA:TTT structure caused by the thymine sugar ring transition, produces 5 degrees bend of the DNA axis directed toward adenines. We also investigated the influence of such conformational disturbance on the neighbouring base pairs, in particular the transition in the trimers with unfrozen ends.     

Structure of the 1,4-bis(2'-deoxyadenosin-N6-yl)-2R,3R-butanediol cross-link arising from alkylation of the human N-ras codon 61 by butadiene diepoxide

The solution structure of the 1,4-bis(2'-deoxyadenosin-N(6)-yl)-2R,3R-butanediol cross-link arising from N(6)-dA alkylation of nearest-neighbor adenines by butadiene diepoxide (BDO(2)) was determined in the oligodeoxynucleotide 5'-d(CGGACXYGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained codon 61 (underlined) of the human N-ras protooncogene. The cross-link was accommodated in the major groove of duplex DNA. At the 5'-side of the cross-link there was a break in Watson-Crick base pairing at base pair X(6).T(17), whereas at the 3'-side of the cross-link at base pair Y(7).T(16), base pairing was intact. Molecular dynamics calculations carried out using a simulated annealing protocol, and restrained by a combination of 338 interproton distance restraints obtained from (1)H NOESY data and 151 torsion angle restraints obtained from (1)H and (31)P COSY data, yielded ensembles of structures with good convergence. Helicoidal analysis indicated an increase in base pair opening at base pair X(6).T(17), accompanied by a shift in the phosphodiester backbone torsion angle beta P5'-O5'-C5'-C4' at nucleotide X(6). The rMD calculations predicted that the DNA helix was not significantly bent by the presence of the four-carbon cross-link. This was corroborated by gel mobility assays of multimers containing nonhydroxylated four-carbon N(6),N(6)-dA cross-links, which did not predict DNA bending. The rMD calculations suggested the presence of hydrogen bonding between the hydroxyl group located on the beta-carbon of the four-carbon cross-link and T(17) O(4), which perhaps stabilized the base pair opening at X(6).T(17) and protected the T(17) imino proton from solvent exchange. The opening of base pair X(6).T(17) altered base stacking patterns at the cross-link site and induced slight unwinding of the DNA duplex. The structural data are interpreted in terms of biochemical data suggesting that this cross-link is bypassed by a variety of DNA polymerases, yet is significantly mutagenic [Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580].