Conformational Variability in Modified Nucleosides. Crystal and Molecular Structure of 2',3'-O-Isopropylidene Inosine

The crystal structure of 2',3'-O-isopropylidene inosine shows a number of interesting features. The four independent molecules in the asymmetric unit exhibit significant conformational variations. Ribose puckers fall in the O(4')-exo region, unfavourable in unsubstituted nucleosides. Hypoxanthine bases show base-pairing (I.I) in a manner analogous to the guanine self pairs (G.G) in 2',3'-O-isopropylidene guanosine but with a C(2)-H…O(6) hydrogen bond instead of N(2)-H…O(6).

Sequence Independent Recombination Triple Helices: A Molecular Dynamics Study

Recent experimental studies have shown that the Rec-A mediated homologous recombination reaction involves a triple helical intermediate, in which the third strand base forms hydrogen bonds with both the bases in the major groove of the Watson-Crick duplex. Such 'mixed' hydrogen bonds allow formation of sequence independent triplexes. DNA triple helices involving 'mixed' hydrogen bonds have been studied, using model building, molecular mechanics (MM) and molecular dynamics (MD). Models were built for a tripler comprising all four possible triplets viz., G.C*C, C.G*G, A.T*T and T.A*A. To check the stability of all the 'mixed' hydrogen bonds in such triplexes and the conformational preferences of such tripler structures, MD studies were carried out starting from two structures with 30 degrees and 36 degrees twist between the basepairs. It was observed that though the two triplexes converged towards a similar structure, the various hydrogen bonds between the WC duplex and the third strand showed differential stabilities. An MD simulation with restrained hydrogen bonds showed that the resulting structure was stable and remained close to the starting structure. These studies help us in defining stable hydrogen bond geometries involving the third strand and the WC duplex. It was observed that in the C.G*G triplets the N7 atom of the second strand is always involved in hydrogen bonding. In the G.C*C triplets, either N3 or O2 in the third strand cytosine can interchangeably act as a hydrogen bond acceptor.

Molecular Mechanics Studies of Homopolymeric and Mixed Sequence Py.Pu*Pu Triple Helices,

DNA triple helices containing two purine strands and one pyrimidine strand (C.G*G and T.A*A) 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 in the C.G*G case the structure with a parallel orientation of the third strand, resulting from Hoogsteen hydrogen bonds between the third strand and the Watson-Crick duplex, is energetically the most favourable while in the T.A*A case the antiparallel orientation of the third strand, resulting from reverse Hoogsteen hydrogen bonds, is energetically the most favourable. These studies when extended to the mixed sequence triplexes, in which the second strand is a mixture of G and A, correspondingly the third strand is a mixture of G and APT, show that though the parallel orientation is still energetically more favourable, the antiparallel orientation becomes energetically comparable with an increasing number of thymines in the third strand. Structurally, for the mixed triplexes containing G and T in the third strand, it is seen that the basepair non-isomorphism between the C.G*G and the T.A*T triplets can be overcome with some changes in the base pair parameters without much distortion of either the backbone or the hydrogen bonds.

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.

Aperiodic Variability in the Cane-Zebiak Model: A Diagnostic Study

The finite predictability of the coupled ocean-atmosphere system is determined by its aperiodic variability. To gain insight regarding the predictability of such a system, a series of diagnostic studies has been carried out to investigate the role of convergence feedback in producing the aperiodic behavior of the standard version of the Cane-Zebiak model. In this model, an increase in sea surface temperature (SST) increases atmospheric heating by enhancing local evaporation (SST anomaly feedback) and low-level convergence (convergence feedback). The convergence feedback is a nonlinear function of the background mean convergence field. For the set of standard parameters used in the model, it is shown that the convergence feedback contributes importantly to the aperiodic behaviour of the model. As the strength of the convergence feedback is increased from zero to its standard value, the model variability goes from a periodic regime to an aperiodic regime through a broadening of the frequency spectrum around the basic periodicity of about 4 years. Examination of the forcing associated with the convergence feedback reveals that it is intermittent, with relatively large amplitude only during 2 or 3 months in the early part of the calendar year. This seasonality in the efficiency of the convergence feedback is related to the strong seasonality of the mean convergence over the eastern Pacific. It is shown that if the mean convergence field is fixed at its March value, aperiodic behavior is produced even in the absence of annual cycles in the other mean fields. On the, other hand, if the mean convergence field is fixed at its September value, the coupled model evolution remains close to periodic, even in the presence of the annual cycle in the other fields. The role of convergence feedback on the aperiodic variability of the model for other parameter regimes is also examined. It is shown that a range exists in the strength of the SST anomaly feedback for which the model variability is aperiodic even without the convergence feedback. It appears that in the absence of convergence feedback, enhancement of the strength of the air-sea coupling in the model through other physical processes also results in aperiodicity in the model.

Both Surface Proteins (VP4 and VP7) of an Asymptomatic Neonatal Rotavirus Strain (1321) Have High Levels of Sequence Identity with the Homologous Proteins of a Serotype 10 Bovine Rotavirus

The nucleotide sequence of genes 4 and 9, encoding the outer capsid proteins VP4 and VP7 of a serotype 10 tissue culture-adapted strain, 1321, representative of asymptomatic neonatal rotaviruses isolated from neonates in Bangalore, India, were determined. Comparison of nucleotide and deduced amino acid sequences of 1321 VP4 and VP7 with previously published sequences of various serotypes revealed that both genes were highly homologous to the respective genes of serotype 10 bovine rotavirus, B223. The VP4 of 1321 represents a new human P serotype and the 1321 and related strains represent the first description of neonatal rotaviruses that appear to derive both surface proteins from an animal rotavirus.

3D MHD simulations of subsurface convection in OB stars

During their main sequence evolution, massive stars can develop convective regions very close to their surface. These regions are caused by an opacity peak associated with iron ionization. Cantiello et al. (2009) found a possible connection between the presence of sub-photospheric convective motions and small scale stochastic velocities in the photosphere of early-type stars. This supports a physical mechanism where microturbulence is caused by waves that are triggered by subsurface convection zones. They further suggest that clumping in the inner parts of the winds of OB stars could be related to subsurface convection, and that the convective layers may also be responsible for stochastic excitation of non-radial pulsations. Furthermore, magnetic fields produced in the iron convection zone could appear at the surface of such massive stars. Therefore subsurface convection could be responsible for the occurrence of observable phenomena such as line profile variability and discrete absorption components. These phenomena have been observed for decades, but still evade a clear theoretical explanation. Here we present preliminary results from 3D MHD simulations of such subsurface convection.

Mechanisms of variability and predictability of the tropical coupled ocean-atmosphere system

A conceptual model is proposed to explain the observed aperiodicity in the short term climate fluctuations of the tropical coupled ocean-atmosphere system. This is based on the evidence presented here that the tropical coupled ocean-atmosphere system sustains a low frequency inter-annual mode and a host of higher frequency intra-seasonal unstable modes. At long wavelengths, the low frequency mode is dominant while at short wavelengths, the high frequency modes are dominant resulting in the co-existence of a long wave low frequency mode with some short wave intra-seasonal modes in the tropical coupled system. It is argued that due to its long wavelength, the low frequency mode would behave like a linear oscillator while the higher frequency short wave modes would be nonlinear. The conceptual model envisages that an interaction between the low frequency linear oscillator and the high frequency nonlinear oscillations results in the observed aperiodicity of the tropical coupled system. This is illustrated by representing the higher frequency intra-seasonal oscillations by a nonlinear low order model which is then coupled to a linear oscillator with a periodicity of four years. The physical mechanism resulting in the aperiodicity in the low frequency oscillations and implications of these results on the predictability of the coupled system are discussed.

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.

Nucleotide sequence of the first cosmid from the Mycobacterium leprae genome project: structure and function of the Rif-Str regions

The nucleotide sequence of cosmid B1790, carrying the Rif-Str regions of the Mycobacterium leprae chromosome, has been determined. Twelve open reading frames were identified in the 36716bp sequence, representing 40% of the coding capacity. Five ribosomal proteins, two elongation factors and the β and β'subunits of RNA polymerase have been characterized and two novel genes were found. One of these encodes a member of the so-called ABC family of ATP-binding proteins while the other appears to encode an enzyme involved in repairing genomic lesions caused by free radicals. This finding may well be significant as M. leprae, an intracellular pathogen, lives within macrophages.

Structure of Sesbania Mosaic Virus at 4·7 Å Resolution and Partial Sequence of the Coat Protein

Sesbania mosaic virus (SMV) is a plant virus infecting Sesbania grandiflora plants in Andhra Pradesh, India. Amino acid sequence of the tryptic peptides of SMV coat protein were determined using a gas phase sequenator. These sequences showed identical amino acids at 69% of the positions when aligned with the corresponding residues of southern bean mosaic virus (SBMV).Crystals diffracting to better than 3 Å resolution were obtained by precipitating the virus with ammonium sulphate. The crystals belonged to rhombohedral space group R3 with α = 291·4 Å and α = 61·9°. Three-dimensional X-ray diffraction data on these crystals were collected to a resolution of 4·7 Å, using a Siemens-Nicolet area detector system. Self-rotation function studies revealed the icosahedral symmetry of the virus particles, as well as their precise orientation in the unit cell. Cross-rotation function and modelling studies with SBMV showed that it is a valid starting model for SMV structure determination. Low resolution phases computed using a polyalanine model of SBMV were subjected to refinement and extension by real-space electron density averaging and solvent flattening. The final electron density map revealed a polypeptide fold similar to SBMV. The single disulphide bridge of SBMV coat protein is retained in SMV. Four icosahedrally independent cation binding sites have been tentatively identified. Three of these sites, related by a quasi threefold axis, are also found in SBMV. The fourth site is situated on the quasi threefold axis. Aspartic acid residues, which replace Ile218 of SBMV from the quasi threefold-related subunits are suitable ligands to the cation at this site

An ensemble of B-DNA dinucleotide geometries lead to characteristic nucleosomal DNA structure and provide plasticity required for gene expression

Background: A nucleosome is the fundamental repeating unit of the eukaryotic chromosome. It has been shown that the positioning of a majority of nucleosomes is primarily controlled by factors other than the intrinsic preference of the DNA sequence. One of the key questions in this context is the role, if any, that can be played by the variability of nucleosomal DNA structure. Results: In this study, we have addressed this question by analysing the variability at the dinucleotide and trinucleotide as well as longer length scales in a dataset of nucleosome X-ray crystal structures. We observe that the nucleosome structure displays remarkable local level structural versatility within the B-DNA family. The nucleosomal DNA also incorporates a large number of kinks. Conclusions: Based on our results, we propose that the local and global level versatility of B-DNA structure may be a significant factor modulating the formation of nucleosomes in the vicinity of high-plasticity genes, and in varying the probability of binding by regulatory proteins. Hence, these factors should be incorporated in the prediction algorithms and there may not be a unique `template' for predicting putative nucleosome sequences. In addition, the multimodal distribution of dinucleotide parameters for some steps and the presence of a large number of kinks in the nucleosomal DNA structure indicate that the linear elastic model, used by several algorithms to predict the energetic cost of nucleosome formation, may lead to incorrect results.

Amino acid selective unlabeling for sequence specific resonance assignments in proteins

Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective `unlabeling' or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly C-13/N-15 labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {(CO)-C-12 (i) -N-15 (i+1)}-filtered HSQC, which aids in linking the H-1(N)/N-15 resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i - 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to H-2 labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of N-14 at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.