Slow motion in the CAA*TTG sequence of a DNA decamer duplex studied by NMR.

In DNA duplexes, pyrimidine-purine steps are believed to be flexible or conformationally unstable. Indeed, several DNA crystal structures exhibit a multitude of conformations for CpA*TpG steps. The question arises of whether this structural flexibility is accompanied by dynamical flexibility, i.e., a question pertaining to the energy barrier between conformations. Except for TpA steps, slow motions on the microsecond-to-millisecond time scale have not been detected in duplexes until now. In the present study, such slow motion was investigated by 1H, 13C, and 15N NMR relaxation measurements on a DNA decamer d(CATTTGCATC)*d(GATGCAAATG). The DNA decamer was enriched with 15% 13C and 98% 15N isotopes for each adenosine and guanosine residue. Three lines of evidence support the notion of slow motion in the CAA*TTG moiety. Analysis of (15)N relaxation showed that the order parameter, S2, of guanosine imino NH groups was about 0.8, similar to that of CH groups for this oligomer. The strong temperature dependence of guanosine NH S2 in the CAA*TTG sequence indicated the presence of a large-amplitude motion. Signals of adenosine H8 protons in the CAA*TTG sequence were broadened in 2D 1H NOESY spectra, which also suggested the existence of slow motion. As well as being smaller than for other adenine residues, the 1H T2 values exhibited a magnetic field strength dependence for all adenosine H8 signals in the ATTTG*CAAAT region, suggesting slow motions more pronounced at the first adenosine in the CAA*TTG sequence but extending over the CAAAT*ATTTG region. This phenomenon was further examined by the pulse field strength dependence of the 1H, 13C, and 15N T1rho values. 1H and 13C T1rho values showed a pulse field strength dependence, but 15N T1rho did not. Assuming a two-site exchange process, an exchange time constant of 20-300 micros was estimated for the first adenosine in the CAA sequence. The exact nature of this motion remains unknown.

Determination of the populations and structures of multiple conformers in an ensemble from NMR data: multiple-copy refinement of nucleic acid structures using floating weights.

A new algorithm is presented for determination of structural conformers and their populations based on NMR data. Restrained Metropolis Monte Carlo simulations or restrained energy minimizations are performed for several copies of a molecule simultaneously. The calculations are restrained with dipolar relaxation rates derived from measured NOE intensities via complete relaxation matrix analysis. The novel feature of the algorithm is that the weights of individual conformers are determined in every refinement step, by the quadratic programming algorithm, in such a way that the restraint energy is minimized. Its design ensures that the calculated populations of the individual conformers are based only on experimental restraints. Presence of internally inconsistent restraints is the driving force for determination of distinct multiple conformers. The method is applied to various simulated test systems. Conformational calculations on nucleic acids are carried out using generalized helical parameters with the program DNAminiCarlo. From different mixtures of A- and B-DNA, minor fractions as low as 10% could be determined with restrained energy minimization. For B-DNA with three local conformers (C2'-endo, O4'-exo, C3'-endo), the minor O4'-exo conformer could not be reliably determined using NOE data typically measured for DNA. The other two conformers, C2'-endo and C3'-endo, could be reproduced by Metropolis Monte Carlo simulated annealing. The behavior of the algorithm in various situations is analyzed, and a number of refinement protocols are discussed. Prior to application of this algorithm to each experimental system, it is suggested that the presence of internal inconsistencies in experimental data be ascertained. In addition, because the performance of the algorithm depends on the type of conformers involved and experimental data available, it is advisable to carry out test calculations with simulated data modeling each experimental system studied.

Conformational dynamics in mixed alpha/beta-oligonucleotides containing polarity reversals: a molecular dynamics study using time-averaged restraints.

Nucleic acid duplexes featuring a single alpha-anomeric thymidine inserted into each DNA strand via 3'-3' and 5'-5' phosphodiester linkages exhibit local conformational dynamics that are not adequately depicted by conventional restrained molecular dynamics (rMD) methods. We have used molecular dynamics with time-averaged NMR restraints (MDtar) to explore its applicability to describing the conformational dynamics of two alpha-containing duplexes--d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2 and d(ATGG-3'-3'-alphaT-5'-5'-GCTC) x r(gagcaccau). In contrast to rMD, enforcing NOE-based distance restraints over a period of time in MDtar rather than instantaneously results in better agreement with the experimental NOE and J-data. This conclusion is based on the dramatic decreases in average distance and coupling constant violations (delta d(av), J(rms), and delta J(av)) and improvements in sixth-root R-factors (R(X)). In both duplexes, the deoxyribose ring puckering behavior predicted independently by pseudorotation analysis is portrayed remarkably well using this approach compared to rMD. This indicates that the local dynamic behavior is encoded within the NOE data, although this is not obvious from the local R(X) values. In both systems, the backbone torsion angles comprising the 3'-3' linkage as well as the (high S-) sugars of the alpha-nucleotide and preceding residue (alpha - 1) are relatively static, while the conformations of the 5'-5' linkage and the sugar in the neighboring beta-nucleotide (alpha + 1) show enhanced flexibility. To reduce the large ensembles generated by MDtar to more manageable clusters we utilized the PDQPRO program. The resulting PDQPRO clusters (in both cases, 13 structures and associated probabilities extracted from a pool of 300 structures) adequately represent the structural and dynamic characteristics predicted by the experimental data.

Solution structure of Syrian hamster prion protein rPrP(90-231).

NMR has been used to refine the structure of Syrian hamster (SHa) prion protein rPrP(90-231), which is commensurate with the infectious protease-resistant core of the scrapie prion protein PrPSc. The structure of rPrP(90-231), refolded to resemble the normal cellular isoform PrPC spectroscopically and immunologically, has been studied using multidimensional NMR; initial results were published [James et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10086-10091]. We now report refinement with better definition revealing important structural and dynamic features which can be related to biological observations pertinent to prion diseases. Structure refinement was based on 2778 unambiguously assigned nuclear Overhauser effect (NOE) connectivities, 297 ambiguous NOE restraints, and 63 scalar coupling constants (3JHNHa). The structure is represented by an ensemble of 25 best-scoring structures from 100 structures calculated using ARIA/X-PLOR and further refined with restrained molecular dynamics using the AMBER 4.1 force field with an explicit shell of water molecules. The rPrP(90-231) structure features a core domain (residues 125-228), with a backbone atomic root-mean-square deviation (RMSD) of 0.67 A, consisting of three alpha-helices (residues 144-154, 172-193, and 200-227) and two short antiparallel beta-strands (residues 129-131 and 161-163). The N-terminus (residues 90-119) is largely unstructured despite some sparse and weak medium-range NOEs implying the existence of bends or turns. The transition region between the core domain and flexible N-terminus, i.e., residues 113-128, consists of hydrophobic residues or glycines and does not adopt any regular secondary structure in aqueous solution. There are about 30 medium- and long-range NOEs within this hydrophobic cluster, so it clearly manifests structure. Multiple discrete conformations are evident, implying the possible existence of one or more metastable states, which may feature in conversion of PrPC to PrPSc. To obtain a more comprehensive picture of rPrP(90-231), dynamics have been studied using amide hydrogen-deuterium exchange and 15N NMR relaxation times (T1 and T2) and 15N{1H} NOE measurements. Comparison of the structure with previous reports suggests sequence-dependent features that may be reflected in a species barrier to prion disease transmission.

A pseudoknot-compatible universal site is located in the large ribosomal RNA in the peptidyltransferase center.

The RNA secondary structure is not confined to a system of the hairpins and can contain pseudoknots as well as topologically equivalent slipped-loop structure (SLS) conformations. A specific primary structure that directs folding to the pseudoknot or SLS is called SL-palindrome (SLP). Using a computer program for searching the SLP in the genomic sequences, 419 primary structures of large ribosomal RNAs from different kingdoms (prokaryota, eukaryota, archaebacteria) as well as plastids and mitochondria were analyzed. A universal site was found in the peptidyltransferase center (PTC) capable of folding to a pseudoknot of 48 nucleotides in length. Phylogenetic conservation of its helices (concurrent replacements with no violation of base pairing, covariation) has been demonstrated. We suggest the reversible folding-unfolding of the pseudoknot for certain stages of the ribosome functioning.

Small structural ensembles for a 17-nucleotide mimic of the tRNA T psi C-loop via fitting dipolar relaxation rates with the quadratic programming algorithm.

Solution structures are typically average structures determined with the help of nmr-derived distance and torsion angle information. However, when a biomolecule populates significantly different conformations, the average structure might be prone to artifacts, and other refinement strategies are necessary. For example, when experimental restraints are used in molecular dynamics simulations in a time-averaged fashion (MDtar), the experimental structural information does no longer need to be satisfied at each step of the simulation; instead, the whole trajectory must agree with the restraints. However, the resulting structural ensembles are large and not unique and it is not trivial to extract the essential dynamic features for a system. Here we demonstrate that large MDtar ensembles can be simplified substantially by reducing the number of members to just a few on the basis of adjusting the individual probabilities of the members with the PDQPRO program [N. B. Ulyanov et al. Biophysical Journal (1995), Vol. 68, p. 13]. This algorithm finds the global minimum for a search function that represents the best match of a given ensemble with the experimental dipolar interproton relaxation rates. We have applied this strategy to a 17-residue RNA hairpin, whose loop exhibited considerable flexibility evident from nmr data. This 17mer is a mimic of the T psi C-loop of tRNA, where nucleotide 54 is usually a ribosylthymidine. The methylation of U54, which is completely buried in transfer ribonucleic acid, is administered by tRNA (m5 U54)-methyltransferase (RUMT). Since the 17mer is a good substrate for RUMT, we previously concluded that the flexibility of the 17mer's loop is a key to how RUMT gains access to the methylation site [L. J. Yao et al. Journal of Biomolecular NMR (1996) Vol. 9. p. 229]. Application of the PDQPRO algorithm to the previously acquired MDtar trajectories allowed us to reduce the number of conformations from several hundred to one major and five or six minor conformations with individual populations from approximately 5% to approximately 50% without any deterioration in the match with the experimental data. The major conformation exhibits a continuation of A-form helicity through part of the loop, involving C60 and U59. In this and most other conformations the methylation site in U54 is no longer buried.

A pseudosquare knot structure of DNA in solution.

We report a high-resolution NMR structure of a homodimer formed by a synthetic 25 residue DNA oligonucleotide GCTCCCATGGTTTTTGTGCACGAGC. This structure presents a novel structural motif for single-stranded nucleic acids, called a pseudosquare knot (PSQ). The oligonucleotide was originally designed to mimic a slipped-loop structure (SLS), another "unusual" DNA structure postulated as an alternative conformation for short direct repeats in double-stranded DNA. The design of the sequence is compatible with both SLS and PSQ structures, both of which possess identical sets of base-paired and unpaired nucleotides but different tertiary folds. We used deuteration of the H8 positions of purines to ascertain that the PSQ is actually formed under the conditions used. The PSQ structure was solved based on homonuclear proton nuclear Overhauser effect data using complete relaxation matrix methods. The structure essentially consists of two side-by-side helices connected by single-stranded loops. Each of the helices is well-defined; however, the relative orientation of the two remains undetermined by the NMR data. The sequences compatible with the PSQ formation are frequent in single-stranded genomes; this structure may play a role as a dimerization motif.

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform.

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of approximately 142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an alpha-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular beta-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

Interproton distance bounds from 2D NOE intensities: effect of experimental noise and peak integration errors.

The effect of experimental and integration errors on the calculation of interproton distances from NOE intensities is examined. It is shown that NOE intensity errors can have a large impact on the distances determined. When multiple spin ('spin diffusion') effects are significant, the calculated distances are often underestimated, even when using a complete relaxation matrix analysis. In this case, the bias of distances to smaller values is due to the random errors in the NOE intensities. We show here that accurate upper and lower bounds of the distances can be obtained if the intensity errors are properly accounted for in the complete relaxation matrix calculations, specifically the MARDIGRAS algorithm. The basic MARDIGRAS algorithm has been previously described [Borgias, B.A. and James, T.L. (1990) J. Magn. Reson., 87, 475-487]. It has been shown to provide reasonably good interproton distance bounds, but experimental errors can compromise the quality of the resulting restraints, especially for weak cross peaks. In a new approach introduced here, termed RANDMARDI (random error MARDIGRAS), errors due to random noise and integration errors are mimicked by the addition of random numbers from within a specified range to each input intensity. Interproton distances are then calculated for the modified intensity set using MARDIGRAS. The distribution of distances that define the upper and lower distance bounds is obtained by using N randomly modified intensity sets. RANDMARDI has been used in the solution structure determination of the interstrand cross-link (XL) formed between 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and the DNA oligomer d(5'-GCGTACGC-3')2 [Spielmann, H.P. et al. (1995) Biochemistry, 34, 12937-12953]. RANDMARDI generates accurate distances bounds from the experimental NOESY cross-peak intensities for the fixed (known) interproton distances in XL. This provides an independent internal check for the ability of RANDMARDI to accurately fit the experimental data. The XL structure determined using RANDMARDI-generated restraints is in good agreement with other biophysical data that indicate that there is no bend introduced into the DNA by the cross-link. In contrast, isolated spin-pair approximation calculations give distance restraints that, when applied in a restrained molecular dynamics protocol, produce a bent structure.

Probability assessment of conformational ensembles: sugar repuckering in a DNA duplex in solution.

Conformational flexibility of molecules in solution implies that different conformers contribute to the NMR signal. This may lead to internal inconsistencies in the 2D NOE-derived interproton distance restraints and to conflict with scalar coupling-based torsion angle restraints. Such inconsistencies have been revealed and analyzed for the DNA octamer GTATAATG.CATATTAC, containing the Pribnow box consensus sequence. A number of subsets of distance restraints were constructed and used in the restrained Monte Carlo refinement of different double-helical conformers. The probabilities of conformers were then calculated by a quadratic programming algorithm, minimizing a relaxation rate-base residual index. The calculated distribution of conformers agrees with the experimental NOE data as an ensemble better than any single structure. A comparison with the results of this procedure, which we term PARSE (Probability Assessment via Relaxation rates of a Structural Ensemble), to an alternative method to generate solution ensembles showed, however, that the detailed multi-conformational description of solution DNA structure remains ambiguous at this stage. Nevertheless, some ensemble properties can be deduced with confidence, the most prominent being a distribution of sugar puckers with minor populations in the N-region and major populations in the S-region. Importantly, such a distribution is in accord with the analysis of independent experimental data--deoxyribose proton-proton scalar coupling constants.

Tertiary base pair interactions in slipped loop-DNA: an NMR and model building study.

Short direct repeat sequences are often found in regulatory regions of various genes; in some cases they display hypersensitivity to S1 nuclease cleavage in supercoiled plasmids. A non-standard DNA structure (Slipped Loop Structure, or SLS) has been proposed for these regions in order to explain the S1 cleavage data; the formation of this structure may be involved in the regulation of transcription. The structure can be generally classified as a particular type of pseudoknot. To date, no detailed stereochemical model has been developed. We have applied one-dimensional 1H NMR spectroscopy to study a synthetic DNA, 55 nucleotides in length, which cannot fold as a standard hairpin but which may favor the SLS formation. AT base pairs were identified, consistent only with the formation of an additional, tertiary miniduplex in the SLS. An all-atom stereochemically sound model has been developed for the SLS with the use of conformational calculations. The model building studies have demonstrated that the tertiary miniduplex can be formed for one of the plausible SLS isomers, but not for the other.

A parallel DNA triplex as a model for the intermediate in homologous recombination.

Parallel DNA triplexes considered to be putative intermediates in homologous recombination, are studied by means of theoretical conformational analysis. These triplexes are denoted as the R-form DNA. Two types of triplexes are analyzed: extended R-form DNA, modeling the triple standard structure, created transiently in the presence of recombination proteins (e.g. RecA); and collapsed R-form, obtained after deproteinization. These structures are stereochemically possible for any arbitrary sequence and have the following properties: (1) the third, R-strand, is parallel to the identical duplex strand and is located in the major groove of the duplex; (2) positions of all four bases in the R-strand are nearly isomorphic; (3) the proposed triplets are consistent with the chemical modification data for deproteinized DNA; we suggest, however, that they are the same in the RecA-DNA complex as well. Since the patterns of charges on each base of the R-strand are strictly complementary to the charges of the homologous Watson-Crick (WC) pair in the major groove, we propose that the selection of the homologous sequence may occur through these complementary electrostatic interactions (electrostatic recognition code). We demonstrate that in the collapsed triplex with a rise of about 3.4 A, the bases from the third R-strand can be inclined and interact with two WC base-pairs simultaneously, which could lead to recognition errors. These mispairings are unlikely in the extended triplex. Therefore, we speculate that a functional role of the extended and underwound DNA structure, transiently formed in the complex with RecA protein, is to obviate such errors and increase the stringency of recognition. In other words, RecA plays the role of a DNA chaperone facilitating the recognition of the single stranded DNA and the duplex. Finally, we show that the proposed isomorphic triplets are conformationally advantageous for strand exchange.

Molecular dynamics with weighted time-averaged restraints for a DNA octamer. Dynamic interpretation of nuclear magnetic resonance data.

For conformationally flexible molecules in solution, NMR-derived distance restraints are time-averaged. In contrast to deriving structures from NMR distance constraints via conventional restrained molecular dynamics (rMD), the range of conformational flexibility may be better represented by MD simulations using weighted time-averaged restrains (MD-tar). This approach has been utilized for dynamic structure refinement of the DNA octamer [d(GTATAATG].[d(CATATTAC)] which contains the Pribnow box consensus sequence. An improved set of distance restraints was calculated via complete relaxation matrix analysis utilizing the solution structure of the octamer which was determined previously by rMD as a starting model. MD-tar calculations were performed with the program AMBER4 under various conditions, some including explicit solvent. All trajectories generated via MD-tar exhibited reduced constraint energies and average deviations for the distances compared to standard rMD. Quantitative comparison with experimental data, i.e. two-dimensional NOE intensities and COSY-derived coupling constants yielded a significant improvement for MD-tar simulated structures relative to rMD-derived structures. The conformational envelope of the MD-tar structures is wider than that from rMD and even unrestrained MD. Explicit solvent force-fields tightened the conformational envelope, leading to even better agreement with experimental data. All MD-tar simulations exhibit sugar repuckering for basically all residues, yielding a minor population in the low N-region and one or more S-conformers. For most backbone torsion angles, one or more minor conformers were found, while the major conformations generally coincided with those obtained in standard rMD. Distributions of helical parameters for MD-tar trajectories are rather symmetric but slightly broader than those for rMD. Average values and associated standard deviations are discussed with respect to sequence-dependent variations. All trajectories obtained with an explicit solvent force field exhibited a narrower minor groove compared to in vacuo calculations.

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: an alternative method for generating a high-resolution solution structure.

A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTATAATG).d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of < 0.3 A between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD < 0.5 A) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.

Decreased interstrand H2-H1' distance in the GC-rich part of the duplex d(CCTCAAACTCC).d(GGAGTTTGAGG) in solution at low temperature: proton nuclear magnetic resonance investigation.

The non-self-complementary undecadeoxyribonucleotide duplex d(CCTCAAACTCC).d(GGAGTTTGAGG) was studied by one- and two-dimensional NMR methods in solution at low and room temperatures. The width of the minor groove of the duplex was determined on the basis of the NOE's between adenine's H2 protons and H1' protons from the complementary strand. In agreement with the previous reports, we found that the A3.T3 block forms a structure with a narrow minor groove at 5 degrees C, with the H2-H1' interstrand distance decreasing in the 5'-to-3' direction along the strand of adenines. Surprisingly, this distance is still short in the GC-rich part of the duplex downstream from the A-tract. This finding is interpreted in terms of pronounced buckle angles in the oligo(purine).oligo(pyrimidine) blocks, which diminish the H2-H1' interstrand distances. Both CA(n)- and AnC- junctions have distinct patterns of the proton chemical shifts, which suggests that both junctions may have some specific conformations in solution. Also, we report the temperature-driven changes in proton chemical shifts, which are significant in all parts of the duplex, except the 5'-ends of both strands. The structural interpretation for these changes is proposed, on the basis of the following notion: at low temperature the narrow minor groove is formed between the central A3.T3 trimer and the 3'-ends of both DNA strands, while the 5'-ends remain relatively exposed to the solvent.

Systematic study of nuclear Overhauser effects vis-à-vis local helical parameters, sugar puckers, and glycosidic torsions in B DNA: insensitivity of NOE to local transitions in B DNA oligonucleotides due to internal structural compensations.

A method has been developed to solve structures of DNA oligomers in solution from the experimental NOE data. The method is a combination of two approaches: (1) full matrix NOESY simulations and (2) conformational calculations of DNA double helix based on generalized helical parameters. The process of the refinement of a solution structure does not involve NMR-derived interproton distance constraints; rather it consists of a direct fitting of a structure to the experimental NOE data, a weighted sum of energy, and R factor being under minimization. A helical parameters-based generation of DNA forms makes it possible to organize the search for the optimal structure more effectively, systematically varying starting conformations. The method has been used to calculate a structure for the self-complementary DNA hexamer GGATCC, which is consistent with the available experimental data. The structure belongs to the B family of forms, although the local structural heterogeneity is very strong. Sugar puckers vary from O4'-exo to C3'-exo; helical steps are open with different magnitudes toward the minor groove. Next, we have addressed the question of how uniquely the structure is defined by the existing NMR data. Different structural parameters have been systematically varied, and their effect on individual NOE's and the R factor has been studied. Two energetically conjugated parameters, sugar puckers and glycosidic angles, can be determined very reliably, because of the strong dependences of the intraresidue H6/H8 to H2'/H2''/H3' NOE's. In contrast, the local helical conformation of DNA and the geometry of base pairs proved to be underdetermined by the existing NOE information, because the effect of any helical parameter on interproton distances can be compensated by the concerted changes in other parameters.

Static and statistical bending of DNA evaluated by Monte Carlo simulations.

To investigate the influence of thermal fluctuations on DNA curvature the Metropolis procedure at 300 K was applied to B-DNA decamers containing A5.T5 and A4.T4 blocks. Monte Carlo simulations have confirmed the DNA bending anisotropy: B-DNA bends most easily in a groove direction (roll). The A5.T5 block is more rigid than the other sequences; the pyrimidine-purine dimers are found to be the most flexible. For A5TCTCT, A5CTCTC, and A5GAGAG, the average bend angle per decamer is 20-25 degrees in a direction toward the minor groove in the center of the A5.T5 tract, which is consistent with both the "junction" and "wedge AA" models. However, in A5T5, A4T4CG, and T4A4GC, bending is directed into the grooves at the 5' and 3' ends of purine tracts. Thus, directionality of bending caused by An.Tn blocks strongly depends on their neighboring sequences. These calculations demonstrate that the sequence-dependent variation of the minor-groove width mimics the observed hydroxyl radical cleavage pattern. To estimate the effect of fluctuations on the overall shape of curved DNA fragments, longer pieces of DNA (up to 200 base pairs) were generated. For sequences with strong curvature (A5X5 and A4T4CG), the static model and Monte Carlo ensemble give similar results but, for moderately and slightly curved sequences (A5T5 or T4A4GC), the static model predicts a much smaller degree of bending than does the statistical representation. Considering fluctuations is important for quantitative interpretation of the gel electrophoresis measurements of DNA curvature, where both the static and statistical bends are operative.

Human c-myc gene contains a regulatory site similar to consensus of interferon response sequence (IRS).

Expression of c-myc proto oncogene is regulated by multiple mechanisms. Here, we report that the consensus of the regulatory region of interferon-dependent genes, GGAAAN1-3 GAAA, was found after computer search in the 5'-terminal flank of human c-myc gene in position (-76:-67). In vitro transcription of c-myc gene fragments showed that the consensus region competes with oligonucleotide GGGAAAATGAAACT for binding to specific protein(s). This oligonucleotide was shown to bind selectively the interferon-dependent positive transcription factor. Transcription of c-myc fragments lacking 5'-terminal region up to positions -101 or +71 was initiated at two sites located in the first intron. These sites did not coincide with P1 in vivo RNA cap-site. Binding of the protein factor(s) to the regulatory region of c-myc gene -76:-67 blocked the in vitro transcription initiated in the first intron.

Sequence-dependent anisotropic flexibility of B-DNA. A conformational study.

Bending flexibility of the six tetrameric duplexes was investigated d(AAAA):d(TTTT), d(AATT)2, d(TTAA)2, d(GGGG):d(CCCC), d(GGCC)2 and d(CCGG)2,. The tetramers were extended in the both directions by regular double helices. The stiffness of the B-DNA double helix when bent into the both grooves proved to be less than that in the perpendicular direction by an order of magnitude. Such an anisotropy is a property of the sugar-phosphate backbone structure. The calculated fluctuations of the DNA bending along the dyad axis, 5-7 degree, are in agreement with experimental value of the DNA persistence length. Anisotropy of the double helix is sequence-dependent: most easily bent into the minor groove are the tetramers with purine-pyrimidine dimer (RY) in the middle. In contrast, YR dinucleotides prefer bending into the major groove. Moreover, they have an equilibrium bend of 6-12 degree into this groove. The above inequality is caused by stacking interaction of the bases. The bend in the central dimer is distributed to some extent between the adjacent links, though the main fraction of the bend remains within the central link. Variation of the sugar-phosphate geometry in the bent helix is inessential, so that DNA remains within the B-family of forms: namely, when the helical axis is bent by 20 degree. the backbone dihedral angles vary by no more than 15 degree. The obtained results are in accord with x-ray structure of the B-DNA dodecamer; they further substantiate our early model of DNA wrapping in the nucleosome by means of "mini-kinks" separated by a half-pitch of the double helix, i.e. by 5-6 b.p. Sequence-dependent anisotropy of DNA presumably dictates the three-dimensional structure of DNA in solution as well. We have found that nonrandom allocation of YR dimers leads to the systematic bends in equilibrium structure of certain DNA fragments.