Probing of unusual DNA structures in topologically constrained form V DNA: use of restriction enzymes as structural probe

The ability of DNA sequences to adopt unusual structures under the superhelical torsional stress has been studied. Sequences that are forced to adopt unusual conformation in topologically constrained pBR322 form V DNA (Lk=0) were mapped using restriction enzymes as probes. Restriction enzymes such as BamHI, Pstl, Aval and HindIII could not cleave their recognition sequences. The removal of topological constraint relieved this inhibition. The influence of neighbouring sequences on the ability of a given sequence to adopt unusual DNA structure, presumably left handed Z conformation, was studied through single hit analysis. Using multiple cut restriction enzymes such as Narl and Fspl, it could be shown that under identical topological strain, the extent of structural alteration is greatly influenced by the neighbouring sequences. In the light of the variety of sequences and locations that could be mapped to adopt non-6 conformation in pBR322 form V DNA, restriction enzymes appear as potential structural probes for natural DNA sequences.

Local variability and base sequence effects in DNA crystal structures.

The importance and usefulness of local doublet parameters in understanding sequence dependent effects has been described for A- and B-DNA oligonucleotide crystal structures. Each of the two sets of local parameters described by us in the NUPARM algorithm, namely the local doublet parameters, calculated with reference to the mean z-axis, and the local helical parameters, calculated with reference to the local helix axis, is sufficient to describe the oligonucleotide structures, with the local helical parameters giving a slightly magnified picture of the variations in the structures. The values of local doublet parameters calculated by NUPARM algorithm are similar to those calculated by NEWHELIX90 program, only if the oligonucleotide fragment is not too distorted. The mean values obtained using all the available data for B-DNA crystals are not significantly different from those obtained when a limited data set is used, consisting only of structures with a data resolution of better than 2.4 A and without any bound drug molecule. Thus the variation observed in the oligonucleotide crystals appears to be independent of the quality of their crystallinity. No strong correlation is seen between any pair of local doublet parameters but the local helical parameters are interrelated by geometric relationships. An interesting feature that emerges from this analysis is that the local rise along the z-axis is highly correlated with the difference in the buckle values of the two basepairs in the doublet, as suggested earlier for the dodecamer structures (Bansal and Bhattacharyya, in Structure & Methods: DNA & RNA, Vol. 3 (Eds., R.H. Sarma and M.H. Sarma), pp. 139-153 (1990)). In fact the local rise values become almost constant for both A- and B-forms, if a correction is applied for the buckling of the basepairs. In B-DNA the AA, AT, TA and GA basepair sequences generally have a smaller local rise (3.25 A) compared to the other sequences (3.4 A) and this seems to be an intrinsic feature of basepair stacking interaction and not related to any other local doublet parameter. The roll angles in B-DNA oligonucleotides have small values (less than +/- 8 degrees), while mean local twist varies from 24 degrees to 45 degrees. The CA/TG doublet sequences show two types of preferred geometries, one with positive roll, small positive slide and reduced twist and another with negative roll, large positive slide and increased twist.(ABSTRACT TRUNCATED AT 400 WORDS)

Circular dichroism and 13C nuclear magnetic resonance spectroscopy of pennisetin from pearl millet

The conformation and stability of pearl millet prolamin (pennisetin) were examined by using circular dichroism and C-13 nuclear magnetic resonance spectroscopy. The far UV spectrum of pennisetin in 70% (v/v) aqueous ethanol showed the presence of predominant alpha-helical structure and its occurrence in the alpha + beta class of protein. The far and near UV spectra of pennisetin in ethanol: trifluoroethanol also supported this observation. However pennisetin showed the presence of some helical structure in 8 M urea which is known to be a highly unordered structure forming solvent. A decrease in alpha helical content of native pennisetin was observed with rise in temperature from 5-75-degrees-C and this effect of temperature was found to be reversible. A C-13 NMR spectrum of pennisetin in 70% ethanol suggested a high degree of molecular mobility in ethanol. Comparison of the cross polarization spectrum with the single pulse excitation spectrum suggested pennisetin to be a heterogeneous protein.

Conformational studies on beta-bend containing a cis peptide unit

Conformational studies have been carried out on the X-cis-Pro tripeptide system (a system of three linked peptide units, in the trans-cis-trans configuration) using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro have been used. The energy minima have been classified into different groups based upon the conformational similarity. There are 15, 20, 18 and 6 minima that are possible for the four cases respectively and these fall into 11 different groups. A study of these minima shows that, (i) some minima contain hydrogen bonds - either 4-->1 or 1-->2 type, (ii) the low energy minima qualify themselves as bend conformations, (iii) cis' and trans' conformations are possible for the prolyl residue as also the C(gamma)-endo and C(gamma)-exo puckerings, and (iv) for Pro-cis-Pro, cis' at the first prolyl residue is ruled out, due to the high energy. The available crystal structure data on proteins and peptides, containing cis-Pro segment have been examined with a view to find the minima that occur in solid state. The data from protein show that they fall under two groups. The conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. In both peptides and proteins there exists a preference for trans' conformation at prolyl residue over cis' when X is a non-glycyl residue. The minima obtained can be useful in modelling studies.

Structural Polymorphism in d(T).d(A)*d(T) Triple Helices

DNA triple helices containing two thymine strands and one adenine strand have been studied, using model building followed by energy minimisation, for different orientations of the third strand resulting from variation in the hydrogen bonding between the Watson-Crick duplex and the third strand and the glycosidic torsion angle in the third strand. Our results show that the structure with a parallel orientation of the third strand, in which the third strand base forms Hoogsteen hydrogen bonds with the adenine base in the Watson-Crick duplex, is energetically the most favourable. An antiparallel orientation of the third strand is also possible, in which the third strand base hydrogen bonds to both the bases in the Watson-Crick duplex. This structure is energetically comparable to the parallel structure. For the parallel triplex a 200ps molecular dynamics simulation starting from two different starting structures indicates that at 300K significant structural heterogeneity exists in this tripler structure. The results are compared with existing structural data on this class of triplexes derived from theoretical and NMR techniques.

Conformational Polymorphism in Telomeric Structures: Loop Orientation and Interloop Pairing in d(G4TnG4),

Sequence repeats constituting the telomeric regions of chromosomes are known to adopt a variety of unusual structures, consisting of a G tetraplex stem and short stretches of thymines or thymines and adenines forming loops over the stem. Detailed model building and molecular mechanics studies have been carried out for these telomeric sequences to elucidate different types of loop orientations and possible conformations of thymines in the loop. The model building studies indicate that a minimum of two thymines have to be interspersed between guanine stretches to form folded-back structures with loops across adjacent strands in a G tetraplex (both over the small as well as large groove), while the minimum number of thymines required to build a loop across the diagonal strands in a G tetraplex is three. For two repeat sequences, these hairpins, resulting from different types of folding, can dimerize in three distinct ways-i.e., with loops across adjacent strands and on same side, with loops across adjacent strands and on opposite sides, and with loops across diagonal strands and on opposite sides-to form hairpin dimer structures. Energy minimization studies indicate that all possible hairpin dimers have very similar total energy values, though different structures are stabilized by different types of interactions. When the two loops are on the same side, in the hairpin dimer structures of d(G(4)T(n)G(4)), the thymines form favorably stacked tetrads in the loop region and there is interloop hydrogen bonding involving two hydrogen bonds for each thymine-thymine pair. Our molecular mechanics calculations on various folded-back as well as parallel tetraplex structures of these telomeric sequences provide a theoretical rationale for the experimentally observed feature that the presence of intervening thymine stretches stabilizes folded-back structures, while isolated stretches of guanines adopt a parallel tetraplex structure

Analysis of sequence dependent variations in secondary and tertiary structure of tRNA molecules

The double helical regions of the five tRNA(Phe) and two tRNA(Asp) crystal structures have been analyzed using the local basepair step parameters. The sequence dependent effects in the mini double helices of tRNA are very similar to those observed in the crystal structures of oligonucleotides in the A-form, the purine-pyrimidine and purine-purine steps have small roll angles when compared to the fiber models of A-DNA as well as A-RNA, while the pyrimidine-purine doublet steps have large roll angles. The orientation of the basepairs in the D-stem is unusual and invariant i.e. they are different from the other three stems but are very similar in all the five tRNA(Phe) crystal structures, presumably due to tertiary interaction of the Watson-Crick basepairs with other bases, with all bases being highly conserved. The origin of the differences between the tertiary structures of tRNA(Phe) and tRNA(Asp) from yeast has also been investigated. It is found that even though the angle between the acceptor arm and the D-stem is very similar in the two structures, the angle subtended by the acceptor arm and the anticodon arm is smaller in the tRNA(Phe) structure (by more than 10 degrees). This is due to differences in the orientation of the two mini helices constituting the anticodon arm, which are inclined to each other by approximately 25 degrees in tRNA(Phe) and 16 degrees in tRNA(Asp). In addition, the acceptor arm, the D-stem and the anticodon stem are nearly coplanar in tRNA(Phe), while in tRNA(Asp) the anticodon stem projects out of the plane defined by the acceptor arm and the anticodon stem. These two features together lead to a larger separation between the acceptor and anticodon ends in tRNA(Asp) and indicate that the junction between the D-stem and the anticodon stem is quite variable, with features characteristic of a ball-and-socket type joint and determined for each tRNA molecule by the base sequence at the junction.

CA/TG sequence at the 5' end of oligo(A)-tracts strongly modulates DNA curvature

An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAACCCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.

Conformational Polymorphism in G-tetraplex Structures: Strand Reversal by Base Flipover or Sugar Flipover

Guanine rich sequences adopt a variety of four stranded structures, which differ in strand orientation and conformation about the glycosidic bond even though they are all stabilised by Hoogsteen hydrogen bonded guanine tetrads. Detailed model building and molecular mechanics calculations have been carried out to investigate various possible conformations of guanines along a strand and different possible orientations of guanine strands in a G-tetraplex structure. It is found that for an oligo G stretch per se, a parallel four stranded structure with all guanines in anti conformation is favoured over other possible tetraplex structures. Hence an alternating syn-anti arrangement of guanines along a strand is likely to occur only in folded back tetraplex structures with antiparallel G strands. Our study provides a theoretical rationale for the observed alternation of glycosidic conformation and the inverted stacking arrangement arising from base flipover, in antiparallel G-tetraplex structures and also highlights the various structural features arising due to different types of strand orientations. The molecular mechanics calculations help in elucidating the various interactions which stabilize different G-tetraplex structures and indicate that screening of phosphate charge by counterions could have a dramatic effect on groove width in these four stranded structures.

Two forms of Pf1 Inovirus: X-ray diffraction studies on a structural phase transition and a calculated libration normal mode of the asymmetric unit

Inovirus is a helical array of alpha-helical protein asymmetric units surrounding a DNA core. X-ray fibre diffraction studies show that the Pf1 species of Inovirus can undergo a reversible temperature-induced transition between two similar structural forms having slightly different virion helix parameters. Molecular models of the two forms show no evidence for altered interactions between the protein and either the solvent or the viral DNA; but there are significant differences in the shape and orientation of the protein asymmetric unit, related to the changes in the virion parameters. Normal modes involving libration of whole asymmetric units are in a frequency range with appreciable entropy of libration, and the structural transition may be related to changes in libration.

Double helix conformation, groove dimensions and ligand binding potential of a G/C stretch in B-DNA

The self-complementary DNA fragment CCGGCGCCGG crystallizes in the rhombohedral space group R3 with unit cell parameters a = 54.07 angstrom and c = 44.59 angstrom. The structure has been determined by X-ray diffraction methods at 2.2 angstrom resolution and refined to an R value of 16.7%. In the crystal, the decamer forms B-DNA double helices with characteristic groove dimensions: compared with B-DNA of random sequence, the minor groove is wide and deep and the major groove is rather shallow. Local base pair geometries and stacking patterns are within the range commonly observed in B-DNA crystal structures. The duplex bears no resemblance to A-form DNA as might have been expected for a sequence with only GC base pairs. The shallow major groove permits an unusual crystal packing pattern with several direct intermolecular hydrogen bonds between phosphate oxygens and cytosine amino groups. In addition, decameric duplexes form quasi-infinite double helices in the crystal by end-to-end stacking. The groove geometries and accessibilities of this molecule as observed in the crystal may be important for the mode of binding of both proteins and drug molecules to G/C stretches in DNA.

Definitions and nomenclature of nucleic acid structure parameters

At an EMBO Workshop on DNA Curvature and Bending, held at Churchill College, Cambridge, on 10-15 September 1988, two sessions were scheduled on definitions of parameters used to describe the geometry of nucleic acid chains and helices, and a common nomenclature for these parameters. The most widely used library of helix analysis programs, HELIB (Fratini et al., 1982; Dickerson, 1985) suffers from the fact that the translations and rotations as defined are not fully independent and depend to a certain extent upon the choice of overall helix axis. Several research groups have been engaged independently in developing alternative programs for the geometrical analysis of polynucleotide chains, but with different definitions of quantities calculated and with widely different nomenclature even when the same parameter was involved.