DNA in profile.

The double helix structure of DNA is not necessarily straight but rather can be curved at almost every base pair. Thus, each piece of DNA possesses a unique silhouette, as individual as its base sequence.

Sequence structure of hidden 10.4-base repeat in the nucleosomes of C. elegans.

By measuring prevailing distances between YY, YR, RR, and RY dinucleotides in the large database of the nucleosome DNA fragments from C. elegans, the consensus sequence structure of the nucleosome DNA repeat of C. elegans was reconstructed: (YYYYYRRRRR)n. An actual period was estimated to be 10.4 bases. The pattern is fully consistent with the nucleosome DNA patterns of other eukaryotes, as established earlier, and, thus, the YYYYYRRRRR repeat can be considered as consensus nucleosome DNA sequence repeat across eukaryotic species. Similar distance analysis for [A, T] dinucleotides suggested the related pattern (TTTYTARAAA)n where the TT and AA dinucleotides display rather out of phase behavior, contrary to the "AA or TT" in-phase periodicity, considered in some publications. A weak 5-base periodicity in the distribution of TA dinucleotides was detected.

Epigenetic nucleosomes: Alu sequences and CG as nucleosome positioning element.

Alu sequences carry periodical pattern with CG dinucleotides (CpG) repeating every 31-32 bases. Similar distances are observed in distribution of DNA curvature in crystallized nucleosomes, at positions +/-1.5 and +/-4.5 periods of DNA from nucleosome DNA dyad. Since CG elements are also found to impart to nucleosomes higher stability when positioned at +/-1.5 sites, it suggests that CG dinucleotides may play a role in modulation of the nucleosome strength when the CG elements are methylated. Thus, Alu sequences may harbor special epigenetic nucleosomes with methylation-dependent regulatory functions. Nucleosome DNA sequence probe is suggested to detect locations of such regulatory nucleosomes in the sequences.

Sequence-directed mapping of nucleosome positions.

A nucleosome DNA sequence probe is designed that combines recently derived RR/YY counter-phase and AA/TT in-phase periodical patterns. A simple nucleosome mapping procedure is introduced for prediction of the nucleosome positions in the sequence of interest, to serve as a guide for experimental studies of the chromatin structure.

Translation framing code and frame-monitoring mechanism as suggested by the analysis of mRNA and 16 S rRNA nucleotide sequences.

Protein coding sequences carry an additional message in the form of a universal three-base periodical pattern (G-non-G-N)n, which is expressed as a strong preference for guanines in the first positions of the codons in mRNA and lack of guanines in the second positions. This periodicity appears immediately after the initiation codon and is maintained along the mRNA as far as the termination triplet, where it disappears abruptly. Known cases of ribosome slippage during translation (leaky frameshifts, out-of-frame gene fusion) are analyzed. At the sites of the slippage the G-periodical pattern is found to be interrupted. It reappears downstream from the slippage sites, in a new frame that corresponds to the new translation frame. This suggests that the (G-non-G-N)n pattern in the mRNA may be responsible for monitoring the correct reading frame during translation. Several sites with complementary C-periodical structure are found in the Escherichia coli 16 S rRNA sequence. Only three of them are exposed to various interactions at the surface of the small ribosomal subunit: (517)gcCagCagCegC, (1395)caCacCgcC and (1531)auCacCucC. A model of a frame-monitoring mechanism is suggested based on the weak complementarity of G-periodical mRNA to the C-periodical sites in the ribosomal RNA. The model is strongly supported by the fact that the hypothetical frame-monitoring sites in the 16 S rRNA that are derived from the nucleotide sequence analysis are also the only sites known to be actually involved or implicated in rRNA-mRNA interactions.

Open and closed 5 S ribosomal RNA, the only two universal structures encoded in the nucleotide sequences.

The nucleotide sequences of small ribosomal RNA (5 S rRNA) molecules of different organisms are presumably designed to ensure folding of these molecules in some standard secondary structure(s). To extract this message contained in the sequences we have plotted the triangular matrix diagrams of all potential hairpins for 44 representative 5 S rRNA sequences. Subjecting these diagrams to simple image-processing procedures we have found that only five hairpins are universally present in all known 5 S rRNA molecules. Two of these hairpins share the same stretch of the nucleotide sequence, others being independent. Thus, only two major secondary structures of 5 S rRNA can be formed. These are of the well-known Y-like (open) form and a novel P-like form closed by the tertiary interaction which involves two complementary stretches four to seven bases long.

Van der Waals locks: loop-n-lock structure of globular proteins.

In a globular protein the polypeptide chain returns to itself many times, making numerous chain-to-chain contacts. The stability of these contacts is maintained primarily by van der Waals interactions. In this work we isolated and analysed van der Waals contacts that stabilise spatial structures of nine major folds. We suggest a specific way to identify the tightest contacts of prime importance for the stability of a given crystallized protein and introduce the notion of the van der Waals lock. The loops closed by the van der Waals interactions provide a basically novel view of protein globule organization: the loop-n-lock structure. This opens a new perspective in understanding protein folding as well: the consecutive looping of the polypeptide chain and the locking of the loop ends by tight van der Waals interactions.

Nucleosome DNA sequence pattern revealed by multiple alignment of experimentally mapped sequences.

Five different algorithms have been applied for detecting DNA sequence pattern hidden in 204 DNA sequences collected from the literature which are experimentally found to be involved in nucleosome formation. Each algorithm was used to perform a multiple alignment of the nucleosome DNA sequences within the window 145 nt, the size of a nucleosome core DNA. From these alignments five pairs of AA and TT dinucleotide positional frequency distributions have been computed. The frequency profiles calculated by different algorithms are rather different due to substantial noise. They, however, share several important features. Both AA and TT dinucleotide positional frequencies display periodicity with the period of 10.3(+/- 0.2) bases. TT dinucleotides appear to be distributed symmetrically relative to AA dinucleotides of the same DNA strand, with the center of symmetry at the midpoint of the nucleosome core DNA. The phase shift between the AA and TT patterns is about 6 bp. Superposition of the five pairs of the AA (TT) positional frequency profiles has produced the refined pattern, with the above features well pronounced. An interesting novel feature of the pattern is an absence of central peaks in the periodical AA and TT distributions. This may indicate that the central section of nucleosome DNA, 15 bp around the dyad axis of the nucleosome, is not bent. Positional distributions of other dinucleotides were not found in this study to be as informative as the ones for AA and TT.

Non-clonability correlates with genomic instability: a case study of a unique DNA region.

Instability of eukaryotic DNA in constructs propagated in prokaryotic hosts is a frequently observed phenomenon. With the exception of a very high A+T-content and the presence of multiple repetitions, no general rule at the basis of this phenomenon is actually known. The intergenic spacer located between the pi and alpha(D) chicken alpha-type globin genes is frequently deleted from recombinant phages and plasmids. Here we have cloned this DNA fragment using a specially designed bacterial strain (SURE competent cells, Stratogene). Comparative analysis of DNA of recombinant clones bearing deletions and clones containing the intact genomic DNA fragment has revealed two important DNA sequence motifs that contribute to the unclonability of eukaryotic DNA in prokaryotic cells. First, the similarity to bacterial transposons (i.e. the presence of repeats flanking a several kilobase DNA fragment) may cause the loss of the fragment during propagation of the recombinant DNA in E. coli. Second, a high content of rotationally correlated kinkable elements (TG*CA steps) may result in non-clonability of the DNA sequence. Interestingly, the latter type of "unclonable" DNA sequence motifs identified in the globin gene domain is unstable (frequently rearranged) also in the eukaryotic chromosome resulting in a local polymorphism. In the chicken domain of alpha globin genes this unstable DNA sequence seems to be partially protected by interaction with nuclear matrix proteins.

Protein sequence modules.

Conserved protein sequence segments are commonly believed to correspond to functional sites in the protein sequence. A novel approach is proposed to profile the changing degree of conservation along the protein sequence, by evaluating the occurrence frequencies of all short oligopeptides of the given sequence in a large proteome database. Thus, a protein sequence conservation profile can be plotted for every protein. The profile indicates where along the sequences the potential functional (conserved) sites are located. The corresponding oligopeptides belonging to the sites are very frequent across many prokaryotic species. Analysis of a representative set of such profiles reveals a common feature of all examined proteins: they consist of sequence modules represented by the peaks of conservation. Typical size of the modules (peak-to-peak distance) is 25-30 amino acid residues.

Consensus temporal order of amino acids and evolution of the triplet code.

Forty different single-factor criteria and multi-factor hypotheses about chronological order of appearance of amino acids in the early evolution are summarized in consensus ranking. All available knowledge and thoughts about origin and evolution of the genetic code are thus combined in a single list where the amino acids are ranked chronologically. Due to consensus nature of the chronology it has several important properties not visible in individual rankings by any of the initial criteria. Nine amino acids of the Miller's imitation of primordial environment are all ranked as topmost (G, A, V, D, E, P, S, L, T). This result does not change even after several criteria related to Miller's data are excluded from calculations. The consensus order of appearance of the 20 amino acids on the evolutionary scene also reveals a unique and strikingly simple chronological organization of 64 codons, that could not be figured out from individual criteria: New codons appear in descending order of their thermostability, as complementary pairs, with the complements recruited sequentially from the codon repertoires of the earlier or simultaneously appearing amino acids. These three rules (Thermostability, Complementarity and Processivity) hold strictly as well as leading position of the earliest amino acids according to Miller. The consensus chronology of amino acids, G/A, V/D, P, S, E/L, T, R, N, K, Q, I, C, H, F, M, Y, W, and the derived temporal order for codons may serve, thus, as a justified working model of choice for further studies on the origin and evolution of the genetic code.

Imported sequences in the mitochondrial yeast genome identified by nucleotide linguistics.

In addition to universally appearing mitochondrial (mt) genes, origins of replication and transcription start regions typical of all mt genome variants of the yeast Saccharomyces cerevisiae, the mt genomes of some of the strains contain variable sequences. These sequences are apparently largely dispensable. They are mainly composed of group-I and -II introns and intergenic open reading frames (ORFs). Many of the introns contain ORFs, some of which were shown by genetic and biochemical means to be involved in splicing and transposition of the mt introns. Some of the optional sequences are hypothesized to be mobile genetic elements. Nucleotide (nt) sequences of the mt genome of S. cerevisiae were examined by analyzing occurrences of oligodeoxyribonucleotide (oligo) 'words'. This linguistic technique had been found to be sensitive to both function and origin of the sequence [Pietrokovski et al., J. Biomol. Struct. Dyn. 7 (1990) 1251-1268]. A clear difference is found between the oligo vocabularies of the optional and basic yeast mt sequences. The difference is mainly located in protein coding segments of the optional sequences which contain conserved amino acid motifs, characteristic of intronic and intergenic ORFs. The use of nt linguistics to detect the sequence dissimilarity and its causes in yeast mitochondria provides fast and straightforward results, identifying the intronic and intergenic ORFs as DNA sequences of foreign, non-mt origin.

Sequence fossils, triplet expansion, and reconstruction of earliest codons.

mRNA sequences are known to carry a hidden periodical pattern (GCU)n, which may be considered a remnant of sequence organization of mRNA early in its evolution, dominated by codons for alanine and their point mutation derivatives. A similar pattern is characteristic of the master (consensus) tRNA sequence derived in 1981 by Eigen and Winkler-Oswatitsch. The master tRNA sequence is thought to represent one of the earliest mRNA. From analysis of literature and from our own calculations presented in this work, the (GCU)n pattern appears to be the most expandable in the norm and in disease. The speculation is put forward that (GCU)n and polyalanine have been key players at the beginning of the triplet code, and the first codons, apart from the GCU triplet, were point change derivatives of the generic triplet GCU, coding for amino acids present in the early prebiotic-biotic environment. The set of the earliest amino acids is derived on the basis of structural simplicity, presence in imitated prebiotic conditions and involvement with class II aminoacyl-tRNA synthetases. The set consists of six amino acids: Ala, Asp, Gly, Pro, Ser and Thr. All these amino acids are, indeed, encoded by the GCU triplet and its derivatives, as predicted. Thus, the pairs GCN (Ala), GAU (Asp), GGU (Gly), CCU (Pro), UCU (Ser) and ACU (Thr) can be viewed as an early triplet code.

Protective nucleosome centering at splice sites as suggested by sequence-directed mapping of the nucleosomes.

The characteristic AA(TT) sequence pattern of the nucleosome DNA derived earlier is used for prediction of nucleosome positions around splice junctions of eukaryotic genes. Two large datasets (2000 sequences each) were collected consisting of DNA segments with the exon/intron and intron/exon splice junctions, from various eukaryotic species. Positions of predicted nucleosomes near the junction sites were calculated. Those junctions which are found to belong to the nucleosomes, are located preferentially within a few base pairs from the midpoint of the nucleosome DNA. That is, obligatory GT- and AG-ends of the introns are more frequently located near the nucleosome dyad axis, within the best protected middle 10-15 base pairs of the nucleosome DNA. In addition, a tendency is observed for the strongest nucleosomes to form more often in the introns, in accordance with the hypothesis on the chromatin-organizing role of introns.

Closed loops of nearly standard size: common basic element of protein structure.

By screening the crystal protein structure database for close Calpha-Calpha contacts, a size distribution of the closed loops is generated. The distribution reveals a maximum at 27+/-5 residues, the same for eukaryotic and prokaryotic proteins. This is apparently a consequence of polymer statistic properties of protein chain trajectory. That is, closure into the loops depends on the flexibility (persistence length) of the chain. The observed preferential loop size is consistent with the theoretical optimal loop closure size. The mapping of the detected unit-size loops on the sequences of major typical folds reveals an almost regular compact consecutive arrangement of the loops. Thus, a novel basic element of protein architecture is discovered; structurally diverse closed loops of the particular size.

Unusual frequencies of certain alternating purine-pyrimidine runs in natural DNA sequences: relation to Z-DNA.

Prokaryotic, eukaryotic and mitochondrial DNA sequences of total Length 300 000 nucleotides have been analyzed to find out whether stretches of alternating purines and pyrimidines are unusual in terms of occurrence, composition and base sequence. Alternating runs longer than 5 nucleotides are significantly under-represented in the natural sequences as compared to random ones. Octanucleotides are the most deficient, occurring at only 60% of the frequency expected in random sequences. An unexpectedly high proportion of these octamers consists of alternating tetramers with the repeat structure (PuPyPuPy)2 or (PyPuPyPu)2. DNA stretches containing such sequences can potentially form a S1 nuclease sensitive slippage (staggered loop) structure, which might serve as a locally unstacked intermediate in the B- to Z-DNA conformational transition.

Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene.

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes.

Recognition of correct reading frame by the ribosome.

The translation frame-monitoring mechanism has been suggested earlier, based on transient complementary contacts, between mRNA and rRNA. Recent studies related to the frame-monitoring mechanism are reviewed. The mechanism is well supported by both new experimental and sequence analysis data. Experiments are suggested for further elucidation of the structural details of the mRNA-rRNA interaction in the ribosome.

Elucidating sequence codes: three codes for evolution.

The sequences are related to evolution in several ways. First, they carry traces of a distant past. Two sequence features point to the earliest sequence organization. The universal hidden GCU-periodical pattern in mRNA suggests the earliest codons: GCU and its nine-point-change derivatives. They code for seven amino acids that by several criteria are also the oldest. Together it makes the earliest form of the triplet code, still recognizable in the extant sequences. Another feature present in the sequences, apparently, since separation of prokaryotes and eukaryotes, is hidden genome segmentation. Both protein-coding and noncoding sequences appear to have been formed by fusion of standard size units, about 360 bp (120 aa) in eukaryotes and 450 bp (150 aa) in prokaryotes. Presumably, the units have been functioning at some stage of evolution as autonomous single-gene size elements. There are sequence designs that promote evolution. One such design suitable for fast adaptation is the tandem repetition of identical sequences, so that their copy numbers in the repeat arrays would modulate (tune) the expression of nearby genes. The tandem repeat expansion diseases illustrate this mechanism in a dramatic way: overtuning of the respective gene expression leads to the disease.

The triplet code from first principles.

Temporal order ("chronology") of appearance of amino acids and their respective codons on evolutionary scene is reconstructed. A consensus chronology of amino acids is built on the basis of 60 different criteria each offering certain temporal order. After several steps of filtering the chronology vectors are averaged resulting in the consensus order: G, A, D, V, P, S, E, (L, T), R, (I, Q, N), H, K, C, F, Y, M, W. It reveals two important features: the amino acids synthesized in imitation experiments of S. Miller appeared first, while the amino acids associated with codon capture events came last. The reconstruction of codon chronology is based on the above consensus temporal order of amino acids, supplemented by the stability and complementarity rules first suggested by M. Eigen and P. Schuster, and on the earlier established processivity rule. At no point in the reconstruction the consensus amino-acid chronology was in conflict with these three rules. The derived genealogy of all 64 codons suggested several important predictions that are confirmed. The reconstruction of the origin and evolutionary history of the triplet code becomes, thus, a powerful research tool for molecular evolution studies, especially in its early stages.