Properties of the nucleic-acid bases in free and Watson-Crick hydrogen-bonded states: computational insights into the sequence-dependent features of double-helical DNA

The nucleic-acid bases carry structural and energetic signatures that contribute to the unique features of genetic sequences. Here we review the connection between the chemical structure of the constituent nucleotides and the polymeric properties of DNA. The sequence-dependent accumulation of charge on the major- and minor-groove edges of the Watson-Crick base pairs, obtained from ab initio calculations, presents unique motifs for direct sequence recognition. The optimization of base interactions generates a propellering of base-pair planes of the same handedness as that found in high-resolution double-helical structures. The optimized base pairs also deform along conformational pathways, i.e., normal modes, of the same type induced by the binding of proteins. Empirical energy computations that incorporate the properties of the base pairs account satisfactorily for general features of the next level of double-helical structure, but miss key sequence-dependent differences in dimeric structure and deformability. The latter discrepancies appear to reflect factors other than intrinsic base-pair structure.     

3DNA: a versatile, integrated software system for the analysis, rebuilding and visualization of three-dimensional nucleic-acid structures

We present a set of protocols showing how to use the 3DNA suite of programs to analyze, rebuild and visualize three-dimensional nucleic-acid structures. The software determines a wide range of conformational parameters, including the identities and rigid-body parameters of interacting bases and base-pair steps, the nucleotides comprising helical fragments, the area of overlap of stacked bases and so on. The reconstruction of three-dimensional structure takes advantage of rigorously defined rigid-body parameters, producing rectangular block representations of the nucleic-acid bases and base pairs and all-atom models with approximate sugar-phosphate backbones. The visualization components create vector-based drawings and scenes that can be rendered as raster-graphics images, allowing for easy generation of publication-quality figures. The utility programs use geometric variables to control the view and scale of an object, for comparison of related structures. The commands run in seconds even for large structures. The software and related information are available at http://3dna.rutgers.edu/.     

Sequence-dependent motions of DNA: a normal mode analysis at the base-pair level

Computer simulation of the dynamic structure of DNA can be carried out at various levels of resolution. Detailed high resolution information about the motions of DNA is typically collected for the atoms in a few turns of double helix. At low resolution, by contrast, the sequence-dependence features of DNA are usually neglected and molecules with thousands of base pairs are treated as ideal elastic rods. The present normal mode analysis of DNA in terms of six base-pair "step" parameters per chain residue addresses the dynamic structure of the double helix at intermediate resolution, i.e., the mesoscopic level of a few hundred base pairs. Sequence-dependent effects are incorporated into the calculations by taking advantage of "knowledge-based" harmonic energy functions deduced from the mean values and dispersion of the base-pair "step" parameters in high-resolution DNA crystal structures. Spatial arrangements sampled along the dominant low frequency modes have end-to-end distances comparable to those of exact polymer models which incorporate all possible chain configurations. The normal mode analysis accounts for the overall bending, i.e., persistence length, of the double helix and shows how known discrepancies in the measured twisting constants of long DNA molecules could originate in the deformability of neighboring base-pair steps. The calculations also reveal how the natural coupling of local conformational variables affects the global motions of DNA. Successful correspondence of the computed stretching modulus with experimental data requires that the DNA base pairs be inclined with respect to the direction of stretching, with chain extension effected by low energy transverse motions that preserve the strong van der Waals' attractions of neighboring base-pair planes. The calculations further show how one can "engineer" the macroscopic properties of DNA in terms of dimer deformability so that polymers which are intrinsically straight in the equilibrium state exhibit the mesoscopic bending anisotropy essential to DNA curvature and loop formation.     

A-form conformational motifs in ligand-bound DNA structures

Recognition and biochemical processing of DNA requires that proteins and other ligands are able to distinguish their DNA binding sites from other parts of the molecule. In addition to the direct recognition elements embedded in the linear sequence of bases (i.e. hydrogen bonding sites), these molecular agents seemingly sense and/or induce an "indirect" conformational response in the DNA base-pairs that facilitates close intermolecular fitting. As part of an effort to decipher this sequence-dependent structural code, we have analyzed the extent of B-->A conformational conversion at individual base-pair steps in protein and drug-bound DNA crystal complexes. We take advantage of a novel structural parameter, the position of the phosphorus atom in the dimer reference frame, as well as other documented measures of local helical structure, e.g. torsion angles, base-pair step parameters. Our analysis pinpoints ligand-induced conformational changes that are difficult to detect from the global perspective used in other studies of DNA structure. The collective data provide new structural details on the conformational pathway connecting A and B-form DNA and illustrate how both proteins and drugs take advantage of the intrinsic conformational mechanics of the double helix. Significantly, the base-pair steps which exhibit pure A-DNA conformations in the crystal complexes follow the scale of A-forming tendencies exhibited by synthetic oligonucleotides in solution and the known polymorphism of synthetic DNA fibers. Moreover, most crystallographic examples of complete B-to-A deformations occur in complexes of DNA with enzymes that perform cutting or sealing operations at the (O3'-P) phosphodiester linkage. The B-->A transformation selectively exposes sugar-phosphate atoms, such as the 3'-oxygen atom, ordinarily buried within the chain backbone for enzymatic attack. The forced remodeling of DNA to the A-form also provides a mechanism for smoothly bending the double helix, for controlling the widths of the major and minor grooves, and for accessing the minor groove edges of individual base-pairs.     

A simple motif for protein recognition in DNA secondary structures

DNA in a single-stranded form (ssDNA) exists transiently within the cell and comprises the telomeres of linear chromosomes and the genomes of some DNA viruses. As with RNA, in the single-stranded state, some DNA sequences are able to fold into complex secondary and tertiary structures that may be recognized by proteins and participate in gene regulation. To better understand how such DNA elements might fold and interact with proteins, and to compare recognition features to those of a structured RNA, we used in vitro selection to identify ssDNAs that bind an RNA-binding peptide from the HIV Rev protein with high affinity and specificity. The large majority of selected binders contain a non-Watson-Crick G.T base-pair and an adjacent C:G base-pair and both are essential for binding. This GT motif can be presented in different DNA contexts, including a nearly perfect duplex and a branched three-helix structure, and appears to be recognized in large part by arginine residues separated by one turn of an alpha-helix. Interestingly, a very similar GT motif is necessary also for protein binding and function of a well-characterized model ssDNA regulatory element from the proenkephalin promoter.     

Elastic network models capture the motions apparent within ensembles of RNA structures

The role of structure and dynamics in mechanisms for RNA becomes increasingly important. Computational approaches using simple dynamics models have been successful at predicting the motions of proteins and are often applied to ribonucleo-protein complexes but have not been thoroughly tested for well-packed nucleic acid structures. In order to characterize a true set of motions, we investigate the apparent motions from 16 ensembles of experimentally determined RNA structures. These indicate a relatively limited set of motions that are captured by a small set of principal components (PCs). These limited motions closely resemble the motions computed from low frequency normal modes from elastic network models (ENMs), either at atomic or coarse-grained resolution. Various ENM model types, parameters, and structure representations are tested here against the experimental RNA structural ensembles, exposing differences between models for proteins and for folded RNAs. Differences in performance are seen, depending on the structure alignment algorithm used to generate PCs, modulating the apparent utility of ENMs but not significantly impacting their ability to generate functional motions. The loss of dynamical information upon coarse-graining is somewhat larger for RNAs than for globular proteins, indicating, perhaps, the lower cooperativity of the less densely packed RNA. However, the RNA structures show less sensitivity to the elastic network model parameters than do proteins. These findings further demonstrate the utility of ENMs and the appropriateness of their application to well-packed RNA-only structures, justifying their use for studying the dynamics of ribonucleo-proteins, such as the ribosome and regulatory RNAs.     

Relationship of DNA structure to internal dynamics: correlation of helical parameters from NOE-based NMR solution structures of d(GCGTACGC)(2) and d(CGCTAGCG)(2) with (13)C order parameters implies conformational coupling in dinucleotide units

The coupling between the conformational properties of double-stranded DNA and its internal dynamics has been examined. The solution structures of the isomeric DNA oligomers d(GCGTACGC)(2) (UM) and d(CGCTAGCG)(2) (CTSYM) were determined with (1)H NMR spectroscopy by utilizing distance restraints from total relaxation matrix analysis of NOESY cross-peak intensities in restrained molecular dynamics calculations. The root-mean-square deviation of the coordinates for the ensemble of structures was 0.13 A for UM and 0.49 A for CTSYM, with crystallographic equivalent R(c)=0.41 and 0.39 and sixth-root residual R(x)=0.11 and 0.10 for UM and CTSYM, respectively. Both UM and CTSYM are B-form with straight helical axes and show sequence-dependent variations in conformation. The internal dynamics of UM and CTSYM were previously determined by analysis of (13)C relaxation parameters in the context of the Lipari & Szabo model-free formalism. Helical parameters for the two DNA oligomers were examined for linear correlations with the order parameters (S(2)) of groups of (13)C spins in base-pairs and dinucleotide units of UM and CTSYM. Correlations were found for six interstrand base-pair parameters tip, y-displacement, inclination, buckle and stretch with various combinations of S(2) for atoms in Watson-Crick base-pairs and for two inter-base-pair parameters, rise and roll with various combinations of S(2) for atoms in dinucleotides. The correlations for the interstrand base-pair helical parameters indicate that the conformations of the deoxyribose residues of each strand are dynamically coupled. Also, the inter-base-pair separation has a profound effect on the local internal motions available to the DNA, supporting the idea that rise is a principal degree of freedom for DNA conformational variability. The correlations indicate collective atomic motions of spins that may represent specific motional modes in DNA, and that base sequence has a predictable effect on the relative order of groups of spins both in the bases and in the deoxyribose ring of the DNA backbone. These observations suggest that an important functional outcome of DNA base sequence is the modulation of both the conformation and dynamic behavior of the DNA backbone.     

Improved RNA secondary structure prediction by maximizing expected pair accuracy

Free energy minimization has been the most popular method for RNA secondary structure prediction for decades. It is based on a set of empirical free energy change parameters derived from experiments using a nearest-neighbor model. In this study, a program, MaxExpect, that predicts RNA secondary structure by maximizing the expected base-pair accuracy, is reported. This approach was first pioneered in the program CONTRAfold, using pair probabilities predicted with a statistical learning method. Here, a partition function calculation that utilizes the free energy change nearest-neighbor parameters is used to predict base-pair probabilities as well as probabilities of nucleotides being single-stranded. MaxExpect predicts both the optimal structure (having highest expected pair accuracy) and suboptimal structures to serve as alternative hypotheses for the structure. Tested on a large database of different types of RNA, the maximum expected accuracy structures are, on average, of higher accuracy than minimum free energy structures. Accuracy is measured by sensitivity, the percentage of known base pairs correctly predicted, and positive predictive value (PPV), the percentage of predicted pairs that are in the known structure. By favoring double-strandedness or single-strandedness, a higher sensitivity or PPV of prediction can be favored, respectively. Using MaxExpect, the average PPV of optimal structure is improved from 66% to 68% at the same sensitivity level (73%) compared with free energy minimization.     

Molecular dynamics simulation reveals conformational switching of water-mediated uracil-cytosine base-pairs in an RNA duplex

A 4 ns molecular dynamics simulation of an RNA duplex (r-GGACUUCGGUCC)(2 )in solution with Na+ and Cl- as counterions was performed. The X-ray structure of this duplex includes two water-mediated uracil-cytosine pairs. In contrast to the other base-pairs in the duplex the water-mediated pairs switch between different conformations. One conformation corresponds to the geometry of the water-mediated UC pairs in the duplex X-ray structure with water acting both as hydrogen-bond donor and acceptor. Another conformation is close to that of a water-mediated UC base-pair found in the X-ray structure of the 23 S rRNA sarcin/ricin domain. In this case the oxygen of the water molecule is linked to two-base donor sites. For a very short time also a direct UC base-pair and a further conformation that is similar to the one found in the RNA duplex structure but exhibits an increased H3(U)...N3(C) distance is observed. Water molecules with unusually long residence times are involved in the water-mediated conformations. These results indicate that the dynamic behaviour of the water-mediated UC base-pairs differs from that of the duplex Watson-Crick and non-canonical guanine-uracil pairs with two or three direct hydrogen bonds. The conformational variability and increased flexibility has to be taken into account when considering these base-pairs as RNA building blocks and as recognition motifs.     

Close correspondence between the motions from principal component analysis of multiple HIV-1 protease structures and elastic network modes

The large number of available HIV-1 protease structures provides a remarkable sampling of conformations of the different conformational states, which can be viewed as direct structural information about the dynamics of the HIV-1 protease. After structure matching, we apply principal component analysis (PCA) to obtain the important apparent motions for both bound and unbound structures. There are significant similarities between the first few key motions and the first few low-frequency normal modes calculated from a static representative structure with an elastic network model (ENM), strongly suggesting that the variations among the observed structures and the corresponding conformational changes are facilitated by the low-frequency, global motions intrinsic to the structure. Similarities are also found when the approach is applied to an NMR ensemble, as well as to molecular dynamics (MD) trajectories. Thus, a sufficiently large number of experimental structures can directly provide important information about protein dynamics, but ENM can also provide similar sampling of conformations.     

Sequence-dependent flexibility in promoter sequences

The non-neighbor interactions between base-pairs were taken into account to calculate the angular parameters (Omega, rho and tau) describing the orientation of successive base-pair planes and the translation parameters (D(y)) along the long axis of base-pair steps for 36 independent tetramers. A statistical mechanical model was proposed to predict the DNA flexibility that is mainly related to the thermal fluctuations at individual base-pair steps. The DNA flexibility can be described by the root-mean-square deviation of the end-to-end distance of DNA helical structure. The present model was then used to investigate the extreme flexible pattern in prokaryotic and eukaryotic promoter sequences. The results demonstrated several extreme flexible regions related to functionally important elements exist both in prokaryotic promoters and in eukaryotic promoters, DNA flexibility and AT content are highly correlated. The probabilities finding flexibility pattern in promoter sequences were also estimated statistically. The biological implications were discussed briefly.     

Influence of sequence on the conformation of the B-DNA helix.

We have tried to ascertain whether the variability found in the conformational features of the 10 base steps in B-DNA is mainly due to the flanking sequences or to interactions with the environment. From an analysis of the twist parameter of the base-pair steps available from crystals of oligonucleotides and protein/oligonucleotide complexes, we conclude that in most cases the flanking sequences show little influence: the conformation of a DNA region results from the combination of the independent intrinsic features of each base step (average conformation and intrinsic variability), modulated by their interactions with the environment. Only in some cases (YR steps, in particular CG and CA/TG) does it appear that flanking sequences have an influence on the conformation of the central base step. The values obtained allow an approximation to the parameters expected for repetitive DNA sequences. In particular, it is found that poly[d(AG/CT)] should have a strongly alternating conformation, in agreement with recently reported oligonucleotide structures.     

A novel roll-and-slide mechanism of DNA folding in chromatin: implications for nucleosome positioning

How eukaryotic genomes encode the folding of DNA into nucleosomes and how this intrinsic organization of chromatin guides biological function are questions of wide interest. The physical basis of nucleosome positioning lies in the sequence-dependent propensity of DNA to adopt the tightly bent configuration imposed by the binding of the histone proteins. Traditionally, only DNA bending and twisting deformations are considered, while the effects of the lateral displacements of adjacent base pairs are neglected. We demonstrate, however, that these displacements have a much more important structural role than ever imagined. Specifically, the lateral Slide deformations observed at sites of local anisotropic bending of DNA define its superhelical trajectory in chromatin. Furthermore, the computed cost of deforming DNA on the nucleosome is sequence-specific: in optimally positioned sequences the most easily deformed base-pair steps (CA:TG and TA) occur at sites of large positive Slide and negative Roll (where the DNA bends into the minor groove). These conclusions rest upon a treatment of DNA that goes beyond the conventional ribbon model, incorporating all essential degrees of freedom of "real" duplexes in the estimation of DNA deformation energies. Indeed, only after lateral Slide displacements are considered are we able to account for the sequence-specific folding of DNA found in nucleosome structures. The close correspondence between the predicted and observed nucleosome locations demonstrates the potential advantage of our "structural" approach in the computer mapping of nucleosome positioning.