Relationship between codon usage and sequence-dependent curvature of genomes

Static DNA curvature distributions of full-sequenced genomes and large DNA contigs from different organisms were calculated. Very distinctive differences among histogram profiles coming from archaebacteria, eubacteria, and eukaryotes were observed. Eubacterial profiles were, on average, more curved than were archaeal and eukaryotic profiles. A comparative analysis between real and randomized DNA sequences revealed that eubacterial genomes presented, overall, higher curvature values than random sequences. An opposite portrait was exhibited by archaeal and eukaryotic genomes. They displayed a lower frequency of curved regions than their corresponding randomized sequences. The contributions of coding and intergenic regions to the curvature profile were also analyzed. Intergenic regions, on average, were found to be more curved than the overall genomic sequences, especially in prokaryotic organisms. Nevertheless, because of their small size with respect to coding regions, the contribution of intergenic sequences to the overall curvature profile tended to be minor. A clear relationship between codon usage and DNA curvature was demonstrated, and a proposal of the possible coevolution of both systems is discussed. Finally, we present a procedure to quantify the deviation of a curvature profile from randomness through a formal statistical analysis.     

Compositional Properties and Thermal Adaptation of SRP-RNA in Bacteria and Archaea

Previous studies have reported a positive correlation between the GC content of the double-stranded regions of structural RNAs and the optimal growth temperature (OGT) in prokaryotes. These observations led to the hypothesis that natural selection favors an increase in GC content to ensure the correct folding and the structural stability of the molecule at high temperature. To date these studies have focused mainly on ribosomal and transfer RNAs. Therefore, we addressed the question of the relationship between GC content and OGT in a different and universally conserved structural RNA, the RNA component of the signal recognition particle (SRP). To this end we generated the secondary structures of SRP-RNAs for mesophilic, thermophilic, and hyperthermophilic bacterial and archaeal species. The analysis of the GC content in the stems and loops of the SRP-RNA of these organisms failed to detect a relationship between the GC contents in the stems of this structural RNA and the growth temperature of bacteria. By contrast, we found that in archaea the GC content in the stem regions of SRP-RNA is highest in hyperthermophiles, intermediate in thermophiles, and lower in mesophiles. In these organisms, we demonstrated a clear positive correlation between the GC content of the stem regions of their SRP-RNAs and their OGT. This correlation was confirmed by a phylogenetic nonindependence analysis. Thus we conclude that in archaea the increase in GC content in the stem regions of SRP-RNA is an adaptation response to environmental temperature.     

Stability and repair of DNA in hyperthermophilic Archaea

Evolutionary and physiological considerations argue that study of hyperthermophilic archaea should reveal new molecular aspects of DNA stabilization and repair. So far, these unusual prokaryotes have yielded a number of genes and enzymatic activities consistent with known mechanisms of excision repair, photo-reversal, and trans-lesion synthesis. However, other DNA enzymes of hyperthermophilic archaea show novel biochemical properties which may be related to DNA stability or repair at extremely high temperature but which remain difficult to evaluate rigorously in vivo. Perhaps the most striking feature of the hyperthermophilic archaea is that all of them whose genomes have been sequenced lack key genes of both the nucleotide excision repair and DNA mismatch repair pathways, which are otherwise highly conserved in biology. Although the growth properties of these micro-organisms hinder experimentation, there is evidence that some systems of excision repair and mutation avoidance operate in Sulfolobus spp. It will therefore be of strategic significance in the next few years to formulate and test hypotheses in Sulfolobus spp. and other hyperthermophilic archaea regarding mechanisms and gene products involved in the repair of UV photoproducts and DNA mismatches.     

A gyrB-like gene from the hyperthermophilic bacterion Thermotoga maritima

We have cloned and sequenced two overlapping DNA fragments (3236 bp) containing a gene encoding the ATPase subunit of a type II DNA topoisomerase from the hyperthermophilic bacterion Thermotoga maritima (Tm Top2B). The deduced protein is composed of 636 aa with a calculated molecular mass of 72 415 Da. It shares significant similarities with the ATPase subunits of mesophilic bacterial DNA topoisomerases II, either DNA gyrase (GyrB) or DNA topoisomerase IV (ParE). Although the highest similarity scores are obtained with GyrB proteins (55% identity with Bacillus subtilis DNA gyrase), a detailed phylogenetic analysis of all known DNA topoisomerases II does not allow us to determine if Tm Top2B corresponds to a DNA gyrase or a DNA topoisomerase IV. This hyperthermophilic Top2B protein exhibits a larger amount of charged amino acids than its mesophilic homologues, a feature which could be important for its thermostability. No gyrA-like gene has been found near top2B. A gene coding for a transaminase B-like protein was found in the upstream region of top2B.     

Congruent evolution of different classes of non-coding DNA in prokaryotic genomes

Prokaryotic genomes are considered to be 'wall-to-wall' genomes, which consist largely of genes for proteins and structural RNAs, with only a small fraction of the genomic DNA allotted to intergenic regions, which are thought to typically contain regulatory signals. The majority of bacterial and archaeal genomes contain 6-14% non-coding DNA. Significant positive correlations were detected between the fraction of non-coding DNA and inter- and intra-operonic distances, suggesting that different classes of non-coding DNA evolve congruently. In contrast, no correlation was found between any of these characteristics of non-coding sequences and the number of genes or genome size. Thus, the non-coding regions and the gene sets in prokaryotes seem to evolve in different regimes. The evolution of non-coding regions appears to be determined primarily by the selective pressure to minimize the amount of non-functional DNA, while maintaining essential regulatory signals, because of which the content of non-coding DNA in different genomes is relatively uniform and intra- and inter-operonic non-coding regions evolve congruently. In contrast, the gene set is optimized for the particular environmental niche of the given microbe, which results in the lack of correlation between the gene number and the characteristics of non-coding regions.     

Stabilization against hyperthermal denaturation through increased CG content can explain the discrepancy between whole genome and 16S rRNA analyses

Based largely upon analysis of ribosomal RNA, a third domain of life, called archaea, had been proposed in addition to bacteria and eukaryotes. However, quantitative analysis of 73 whole genomes shows only a two-domain division of life: into eukaryotes and prokaryotes. Thousands of orthologous genes in archaea and bacteria show an essentially unimodal distribution of sequence identities. Thus, whole genome analyses indicate that archaea are a phylum of bacteria rather than a separate domain of life. In contrast, archaeal rRNA and that of hyperthermophilic bacteria differ from the rRNA of mesophilic bacteria. Thus, there is a bimodal distribution of rRNA sequence identities which differ by 12%. This discrepancy in rRNA and gene content based analyses of whole genomes is likely due to a 15% elevated C:G content of the rRNA of archaea and hyperthermophilic bacteria. The elevated C:G content is consistent with stabilization against thermal denaturation caused by additional hydrogen bonding (3 bonds) in C:G pairs compared to A:U pairs (2 bonds). Based upon this premise, there is no reliable way to correct rRNA for such differences in base composition and it is not possible to quantitatively compare hyperthermophiles with mesophiles by the rRNA method. Furthermore, quantitative study of whole genomes shows that the extent of change in both bacterial and archaeal genes, including rRNA, has reached a limit. Thus, direct sequence comparisons work with closely related genomes, but it is not possible to differentiate the most divergent prokaryotic species, which are currently designated as separate phyla. We believe that the differences in characteristics of archaeal species is based primarily upon selection of genes and pathways compatible with the extreme environmental lifestyle, i.e., hyperthermophily.     

极端嗜热古菌DNA修复核酸内切酶的研究进展

极端嗜热古菌由于生活在高温环境,其基因组DNA面临着严重的挑战,因此,它们如何维持其基因组稳定是本研究领域最为关注的科学问题之一。极端嗜热古菌具有与常温微生物相似的自发突变频率,暗示着它们比常温微生物具有更加有效的DNA修复体系进行修复高温所造成的基因组DNA损伤。目前,极端嗜热古菌DNA修复的分子机制尚不清楚。核酸内切酶在DNA修复途径中发挥着重要的作用。基因组序列显示极端嗜热古菌编码多种DNA修复核酸内切酶,但是其研究尚处于初期阶段。本文综述了极端嗜热古菌DNA修复核酸内切酶Nuc S、Endo V、Endo Q、XPF和Hjc的研究进展,并对今后的研究提出了展望。     

Automatic discovery of regulatory patterns in promoter regions based on whole cell expression data and functional annotation

MOTIVATION: The whole genomes submitted to GenBank contain valuable information about the function of genes as well as the upstream sequences and whole cell expression provides valuable information on gene regulation. To utilize these large amounts of data for a biological understanding of the regulation of gene expression, new automatic methods for pattern finding are needed. RESULTS: Two word-analysis algorithms for automatic discovery of regulatory sequence elements have been developed. We show that sequence patterns correlated to whole cell expression data can be found using Kolmogorov-Smirnov tests on the raw data, thereby eliminating the need for clustering co-regulated genes. Regulatory elements have also been identified by systematic calculations of the significance of correlations between words found in the functional annotation of genes and DNA words occurring in their promoter regions. Application of these algorithms to the Saccharomyces cerevisiae genome and publicly available DNA array data sets revealed a highly conserved 9-mer occurring in the upstream regions of genes coding for proteasomal subunits. Several other putative and known regulatory elements were also found. AVAILABILITY: Upon request.     

A non-curved chicken lysozyme 5'' matrix attachment site is 3'' followed by a strongly curved DNA sequence.

Matrix attachment regions (MARs) partition the genome into functional and structural loop-domains. Here, we determined the relative matrix affinity of cloned fragments of the chicken lysozyme 5' MAR. We show that this region contains a non-curved high-affinity binding site, which is 3' followed by a strongly curved DNA sequence that exhibits weak matrix binding. DNA curvature is not a physical property required for strong matrix binding. Possible biological functions of this sequence arrangement, particularly of the strongly curved DNA, are discussed.     

Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability.

Understanding the molecular determinants of protein thermostability is of theoretical and practical importance. While numerous determinants have been suggested, no molecular feature has been judged of paramount importance, with the possible exception of ion-pair networks. The difficulty in identifying the main determinants may have been the limited structural information available on the thermostable proteins. Recently the complete genomes for mesophilic, thermophilic and hyperthermophilic organisms have been sequenced, vastly improving the potential for uncovering general trends in sequence and structure evolution related to thermostability and, thus, for isolating the more important determinants. From a comparative analysis of 20 complete genomes, we find a trend towards shortened thermophilic proteins relative to their mesophilic homologs. Moreover, sequence alignments to proteins of known structure indicate that thermophilic sequences are more likely than their mesophilic homologs to have deletions in exposed loop regions. The new genomes offer enough comparable sequences to compute meaningful statistics that point to loop deletion as a general evolutionary strategy for increasing thermostability.     

Role of curved DNA in binding of Escherichia coli RNA polymerase to promoters.

The ability of curved DNA upstream of the -35 region to affect the interaction of Escherichia coli RNA polymerase and promoter DNA was examined through the use of hybrid promoters. These promoters were constructed by substituting the curved DNA from two Bacillus subtilis bacteriophage SP82 promoters for the comparable DNA of the bacteriophage lambda promoters lambda pR and lambda pL. The SP82 promoters possessed intrinsic DNA curvature upstream of their -35 regions, as characterized by runs of adenines in phase with the helical repeat. In vitro, the relative affinities of purified sigma 70-RNA polymerase for the promoters were determined in a competition binding assay. Hybrid promoters derived from lambda pR that contained curved DNA were bound by E. coli RNA polymerase more efficiently than was the original lambda pR. Binding of E. coli RNA polymerase to these hybrid promoters was favored on superhelical DNA templates according to gel retardation analysis. Both the supercoiled and relaxed forms of the hybrid lambda pL series were better competitors for E. coli RNA polymerase binding than was the original lambda pL. The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid lambda pR promoters around the E. coli RNA polymerase in a tight, nucleosomal-like fashion. The tight wrapping of the upstream DNA around the polymerase may facilitate the subsequent steps of DNA untwisting and strand separation.     

A Possible Role of DNA Superstructures in Genome Evolution

The concept of DNA as a simple repository of the gene information has changed in that of a polymorphic macromolecule, which plays a relevant part in the management of the complex biochemical transformations in living matter. As a consequence of the slight stereochemical differences between base pairs, the direction of the DNA double helix axis undergoes deterministic writhing. A useful representation of such sequence-dependent structural distortions is the curvature diagram. Here, it is reported as an evolution simulation obtained by extensive point mutations along a biologically important DNA tract. The curvature changes, consequence of the point mutations. were compared to the related experimental gel electrophoresis mobility. The curvature of most mutants decreases and the mobility increases accordingly, suggesting the curvature of that tract is genetically selected. Moreover, DNA images by scanning force microscopy, show evidence of a sequence-dependent adhesion of curved DNA tracts to inorganic crystal surfaces. In particular, mica shows a large affinity towards the TT-rich dinucleotide sequences. This suggests a possible mechanism of selection of curved DNA regions, characterized by AA.TT dinucleotides in phase with double-helical periodicity, in the very early evolution steps.     

High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes

The causes of the variation between genomes in their guanine (G) and cytosine (C) content is one of the central issues in evolutionary genomics. The thermal adaptation hypothesis conjectures that, as G:C pairs in DNA are more thermally stable than adenonine:thymine pairs, high GC content may he a selective response to high temperature. A compilation of data on genomic GC content and optimal growth temperature for numerous prokaryotes failed to demonstrate the predicted correlation. By contrast, the GC content of Structural RNAs is higher at high temperatures. The issue that we address here is whether more freely evolving sites in exons (i.e. codonic third positions) evolve in the same manner as genomic DNA as a whole, Showing no correlated response, or like structural RNAs showing a strong correlation. The latter pattern would provide strong support for the thermal adaptation hypothesis, as the variation in GC content between orthologous genes is typically most profoundly seen at codon third sites (GC3). Simple analysis of completely sequenced prokaryotic genomes shows that GC3, but not genomic GC, is higher on average in thermophilic species. This demonstrates, if nothing else, that the results from the two measures cannot be presumed to be the same. A proper analysis, however, requires phylogenetic control. Here, therefore, we report the results of a comparative analysis of GC composition and optimal growth temperature for over 100 prokaryotes. Comparative analysis fails to show, in either Archea or Eubacteria, any hint of connection between optimal growth temperature and GC content in the genome as a whole, in protein-coding regions or, more crucially at GC. Conversely, comparable analysis confirms that GC content of structural RNA is strongly correlated with optimal temperature. Against the expectations of the thermal adaptation hypothesis, within prokaryotes GC content in protein-coding genies, even at relatively freely evolving sites, cannot be considered an adaptation to the thermal environment.