Sequence and structural conservation in RNA ribose zippers

The "ribose zipper", an important element of RNA tertiary structure, is characterized by consecutive hydrogen-bonding interactions between ribose 2'-hydroxyls from different regions of an RNA chain or between RNA chains. These tertiary contacts have previously been observed to also involve base-backbone and base-base interactions (A-minor type). We searched for ribose zipper tertiary interactions in the crystal structures of the large ribosomal subunit RNAs of Haloarcula marismortui and Deinococcus radiodurans, and the small ribosomal subunit RNA of Thermus thermophilus and identified a total of 97 ribose zippers. Of these, 20 were found in T. thermophilus 16 S rRNA, 44 in H. marismortui 23 S rRNA (plus 2 bridging 5 S and 23 S rRNAs) and 30 in D. radiodurans 23 S rRNA (plus 1 bridging 5 S and 23 S rRNAs). These were analyzed in terms of sequence conservation, structural conservation and stability, location in secondary structure, and phylogenetic conservation. Eleven types of ribose zippers were defined based on ribose-base interactions. Of these 11, seven were observed in the ribosomal RNAs. The most common of these is the canonical ribose zipper, originally observed in the P4-P6 group I intron fragment. All ribose zippers were formed by antiparallel chain interactions and only a single example extended beyond two residues, forming an overlapping ribose zipper of three consecutive residues near the small subunit A-site. Almost all ribose zippers link stem (Watson-Crick duplex) or stem-like (base-paired), with loop (external, internal, or junction) chain segments. About two-thirds of the observed ribose zippers interact with ribosomal proteins. Most of these ribosomal proteins bridge the ribose zipper chain segments with basic amino acid residues hydrogen bonding to the RNA backbone. Proteins involved in crucial ribosome function and in early stages of ribosomal assembly also stabilize ribose zipper interactions. All ribose zippers show strong sequence conservation both within these three ribosomal RNA structures and in a large database of aligned prokaryotic sequences. The physical basis of the sequence conservation is stacked base triples formed between consecutive base-pairs on the stem or stem-like segment with bases (often adenines) from the loop-side segment. These triples have previously been characterized as Type I and Type II A-minor motifs and are stabilized by base-base and base-ribose hydrogen bonds. The sequence and structure conservation of ribose zippers can be directly used in tertiary structure prediction and may have applications in molecular modeling and design.     

Sequence and structural determinants of mannose recognition

Mannose, an abundant cell surface monosaccharide binds to a diverse set of receptors, which are involved in a variety of important cellular processes. Structural analysis has been carried out on all the proteins containing non-covalently bound mannose as a monosaccharide in the Protein Data Bank, to identify common recognition principles. Proteins, highly specific to mannose, belonging to the super family of bulb lectins, are found to contain a consensus sequence motif QXDXNXVXY, which has been identified to be essential for mannose binding. Analysis of this motif in the crystal structures of bulb lectins has led to the understanding of the contribution of individual residues in mannose recognition. Comparison with other mannose binding proteins, reveals common hydrogen bonding patterns in all of them, despite differences in sequence, overall fold and the substructures at the binding sites of individual proteins. A database analysis also suggests that although the topology of the backbone, as at the binding site in bulb lectins, can generate mannose binding capability in a few other proteins, sequence and disposition of not only the residues in the motif, but also the residues in the neighborhood play a crucial role in allowing that property to be retained.     

Sequence diversity, reproductive isolation and species concepts in Saccharomyces

Using the biological species definition, yeasts of the genus Saccharomyces sensu stricto comprise six species and one natural hybrid. Previous work has shown that reproductive isolation between the species is due primarily to sequence divergence acted upon by the mismatch repair system and not due to major gene differences or chromosomal rearrangements. Sequence divergence through mismatch repair has also been shown to cause partial reproductive isolation among populations within a species. We have surveyed sequence variation in populations of Saccharomyces sensu stricto yeasts and measured meiotic sterility in hybrids. This allows us to determine the divergence necessary to produce the reproductive isolation seen among species. Rather than a sharp transition from fertility to sterility, which may have been expected, we find a smooth monotonic relationship between diversity and reproductive isolation, even as far as the well-accepted designations of S. paradoxus and S. cerevisiae as distinct species. Furthermore, we show that one species of Saccharomyces--S. cariocanus--differs from a population of S. paradoxus by four translocations, but not by sequence. There is molecular evidence of recent introgression from S. cerevisiae into the European population of S. paradoxus, supporting the idea that in nature the boundary between these species is fuzzy.     

Sequence specificity in spermine-induced structural changes in CG-oligomers

The role of spermine in inducing A-DNA conformation in deoxyoligonucleotides has been studied using CCGG and GGCC as model sequences. It has been found that while CCGG adopts an alternating B-DNA conformation in low salt solution at low temperature, addition of spermine to this medium induces a B --greater than A transition. In contrast, the A-DNA-like structure of GGCC in low salt solution at low temperature does not change under the influence of spermine. This suggests a sequence-dependent behaviour of spermine. Further these results suggest that the A-DNA conformation observed in the crystals of d(iCCGG) and d(GGCC)2 might have been due to the presence of spermine in the crystallization cocktail.     

Sequence evolution correlates with structural dynamics

Biochemical activity and core stability are essential properties of proteins, maintained usually by conserved amino acids. Structural dynamics emerged in recent years as another essential aspect of protein functionality. Structural dynamics enable the adaptation of the protein to binding substrates and to undergo allosteric transitions, while maintaining the native fold. Key residues that mediate structural dynamics would thus be expected to be conserved or exhibit coevolutionary patterns at least. Yet, the correlation between sequence evolution and structural dynamics is yet to be established. With recent advances in efficient characterization of structural dynamics, we are now in a position to perform a systematic analysis. In the present study, a set of 34 enzymes representing various folds and functional classes is analyzed using information theory and elastic network models. Our analysis shows that the structural regions distinguished by their coevolution propensity as well as high mobility are predisposed to serve as substrate recognition sites, whereas residues acting as global hinges during collective dynamics are often supported by conserved residues. We propose a mobility scale for different types of amino acids, which tends to vary inversely with amino acid conservation. Our findings suggest the balance between physical adaptability (enabled by structure-encoded motions) and chemical specificity (conferred by correlated amino acid substitutions) underlies the selection of a relatively small set of versatile folds by proteins.     

Sequence and diversity of the rat delta T-cell receptor

The cDNA sequence of the delta T-cell receptor (TCRD) in the adult Lewis rat thymus was determined using the technique of rapid amplification of cDNA ends. Sixteen variable region genes (TCRDV), two diversity regions (TCRDD), two joining regions (TCRDJ), and a single constant region gene (TCRDC) were identified. The sixteen unique TCRDV genes identified represented eight different subfamilies in the rat and were highly conserved (>80% nucleotide identity) to corresponding mouse sequences. Extensive junctional diversity was observed in the rat, with both TCRDD regions (TCRDD1 and TCRDD2) utilized in the majority of cDNA clones identified. The two TCRDJ genes were highly conserved and corresponded to TCRDJ1 and TCRDJ2 in the mouse; the majority of clones utilized TCRDJ1. The TCRDC region in the rat was 91.1% identical to the mouse TCRDC gene and was highly conserved to other species. Although extensive sequence information about mouse gamma-delta T-cell receptor genes is available, current knowledge of rat gamma-delta T-cells is limited. The sequence analysis presented in this study adds to our understanding of gamma-delta T-cells in general, and it may be utilized to study the role of gamma-delta T-cells in immune-mediated disease and transplantation models previously established in the rat.     

Fish microsporidia: fine structural diversity and phylogeny

Structural diversity of fish microsporidian life cycle stages and of the host-parasite interface is reviewed. In the infected cell of the fish host, microsporidia may either cause serious degradation of the cytoplasm and demise of the cell, or they may elicit host cell hypertrophy, producing a parasite-hypertrophic host cell complex, the xenoma. The structure of the xenoma and of its cell wall may differ according to the genus of the parasite, and seems to express properties of the parasite rather than those of the host. In merogony, the parasite cell surface interacts with the host cell in diverse ways, the most conspicuous being the production of thick envelopes of different types. Sporogony stages reveal different types of walls or membranes encasing the sporoblasts and later the spores and these envelopes may be of host or parasite origin. Nucleospora differs from all other fish microsporidia by its unique process of sporogony. Except for the formation of conspicuous xenomas, there are no essentially different structures in fish-infecting microsporidia compared with microsporidia from other hosts. Although the structures associated with the development of fish microsporidia cannot be attributed importance in tracing the phylogeny, they are relevant for practical determination and assessing the relation to the host. The possibility of the existence of an intermediate host is discussed. Higher-level classification of Microsporidia is briefly discussed and structure and evolutionary rates in microsporidian rDNA are reviewed. Discussion of rDNA molecular phylogeny of fish-infecting microsporidia is followed by classification of these parasites. Most form a rather cohesive clade. Outside this clade is the genus Nucleospora, separated at least at the level of Order. Within the main clade, however, there are six species infecting hosts other than fish. Based on data available for analysis, a tentative classification of fish-infecting microsporidia into five groups is proposed. Morphologically defined groups represent families, others are referred to as clades. Group 1, represented by family Pleistophoridae, includes Pleistophora, Ovipleistophora and Heterosporis; Vavraia and Trachipleistophora infect non-fish hosts. Group 2, represented by family Glugeidae, is restricted to genus Glugea and Tuzetia weidneri from crustaceans. Group 3 comprises three clades: Loma and a hyperparasitic microsporidian from a myxosporean; Ichthyosporidium and Pseudoloma clade and the Loma acerinae clade. For the latter species a new genus has to be established. Group 4 contains two families, Spragueidae with the genus Spraguea and Tetramicridae with genera Microgemma and Tetramicra, and the Kabatana and Microsporidium seriolae clade. Group 5 is represented by the family Enterocytozoonidae with the genus Nucleospora and mammal-infecting genus Enterocytozoon.     

Sequence and structural differences between enzyme and nonenzyme homologs

To improve our understanding of the evolution of novel functions, we performed a sequence, structural, and functional analysis of homologous enzymes and nonenzymes of known three-dimensional structure. In most examples identified, the nonenzyme is derived from an ancestral catalytic precursor (as opposed to the reverse evolutionary scenario, nonenzyme to enzyme), and the active site pocket has been disrupted in some way, owing to the substitution of critical catalytic residues and/or steric interactions that impede substrate binding and catalysis. Pairwise sequence identity is typically insignificant, and almost one-half of the enzyme and nonenzyme pairs do not share any similarity in function. Heterooligomeric enzymes comprising homologous subunits in which one chain is catalytically inactive and enzyme polypeptides that contain internal catalytic and noncatalytic duplications of an ancient enzyme domain are also discussed.     

Beta propellers: structural rigidity and functional diversity

Recently solved structures and proposed models have helped to reveal the structural characteristics of the beta-propeller fold, as well as the features that contribute to its high rigidity and stability. Possible strategies for identifying beta-propeller proteins in newly characterised sequences are helping to overcome the problems of predicting the beta-propeller fold from amino acid sequences.     

Sequence and diversity of rabbit T-cell receptor gamma chain genes

The nucleotide sequences of one constant (C), six variable (V), and two joining (J) gene segments coding for the rabbit T-cell receptor gamma chain (Tcrg) were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction. The Tcrg-C gene segment did not encode a cysteine residue for connection to the Tcr delta chain in the connecting region, and two variant forms of the Tcrg-C gene segment were generated by alternative splicing, like the human Tcrg-C2 gene. Five of six rabbit Tcrg-V gene segments belonged to the same family and displayed similarity to five productive human Tcrg-V1 family genes as well as the mouse Tcrg-V5 gene. The remaining rabbit Tcrg-V gene segment displayed similarity to the human Tcrg-V3 gene. Both rabbit Tcrg-J gene segments displayed similarity to the human Tcrg-J2.1 and 2.3, respectively. These findings suggested that the genomic organization of rabbit Tcrg genes is more similar to that of human than of mouse Tcrg genes.The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases and have been assigned the accession numbers D38134-D38144 and D42090     

Sequence and structural selectivity of nucleic acid binding ligands

The sequence and structural selectivity of 15 different DNA binding agents was explored using a novel, thermodynamically rigorous, competition dialysis procedure. In the competition dialysis method, 13 different nucleic acid structures were dialyzed against a common ligand solution. More ligand accumulated in the dialysis tube containing the structural form with the highest ligand binding affinity. DNA structural forms included in the assay ranged from single-stranded forms, through a variety of duplex forms, to multistranded triplex and tetraplex forms. Left-handed Z-DNA, RNA, and a DNA-RNA hybrid were also represented. Standard intercalators (ethidium, daunorubicin, and actinomycin D) served as control compounds and were found to show structural binding preferences fully consistent with their previously published behavior. Standard groove binding agents (DAPI, distamycin, and netropsin) showed a strong preference for AT-rich duplex DNA forms, along with apparently strong binding to the poly(dA)-[poly(dT)](2) triplex. Thermal denaturation studies revealed the apparent triplex binding to be complex, and perhaps to result from displacement of the third strand. Putative triplex (BePI, coralyne, and berberine) and tetraplex [H(2)TmPyP, 5,10,15, 20-tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine, and N-methyl mesoporphyrin IX] selective agents showed in many cases less dramatic binding selectivity than anticipated from published reports that compared their binding to only a few structural forms. Coralyne was found to bind strongly to single-stranded poly(dA), a novel and previously unreported interaction. Finally, three compounds (berenil, chromomycin A, and pyrenemethylamine) whose structural preferences are largely unknown were examined. Pyrenemethylamine exhibited an unexpected and unprecedented preference for duplex poly(dAdT).     

Sequence diversity in 36 candidate genes for cardiovascular disorders.

Two strategies involving whole-genome association studies have been proposed for the identification of genes involved in complex diseases. The first one seeks to characterize all common variants of human genes and to test their association with disease. The second one seeks to develop dense maps of single-nucleotide polymorphisms (SNPs) and to detect susceptibility genes through linkage disequilibrium. We performed a molecular screening of the coding and/or flanking regions of 36 candidate genes for cardiovascular diseases. All polymorphisms identified by this screening were further genotyped in 750 subjects of European descent. In the whole set of genes, the lengths explored spanned 53.8 kb in the 5' regions, 68.4 kb in exonic regions, and 13 kb in the 3' regions. The strength of linkage disequilibrium within candidate regions suggests that genomewide maps of SNPs might be efficient ways to identify new disease-susceptibility genes, provided that the maps are sufficiently dense. However, the relatively large number of polymorphisms within coding and regulatory regions of candidate genes raises the possibility that several of them might be functional and that the pattern of genotype-phenotype association might be more complex than initially envisaged, as actually has been observed in some well-characterized genes. These results argue in favor of both genomewide association studies and detailed studies of the overall sequence variation of candidate genes, as complementary approaches.     

The structural diversity of artificial genetic polymers

Synthetic genetics is a subdiscipline of synthetic biology that aims to develop artificial genetic polymers (also referred to as xeno-nucleic acids or XNAs) that can replicate in vitro and eventually in model cellular organisms. This field of science combines organic chemistry with polymerase engineering to create alternative forms of DNA that can store genetic information and evolve in response to external stimuli. Practitioners of synthetic genetics postulate that XNA could be used to safeguard synthetic biology organisms by storing genetic information in orthogonal chromosomes. XNA polymers are also under active investigation as a source of nuclease resistant affinity reagents (aptamers) and catalysts (xenozymes) with practical applications in disease diagnosis and treatment. In this review, we provide a structural perspective on known antiparallel duplex structures in which at least one strand of the Watson–Crick duplex is composed entirely of XNA. Currently, only a handful of XNA structures have been archived in the Protein Data Bank as compared to the more than 100 000 structures that are now available. Given the growing interest in xenobiology projects, we chose to compare the structural features of XNA polymers and discuss their potential to access new regions of nucleic acid fold space.     

Mycobacterial adenylyl cyclases: biochemical diversity and structural plasticity

The conversion of adenine and guanine nucleoside triphosphates to cAMP and cGMP is carried out by nucleotide cyclases, which vary in their primary sequence and are therefore grouped into six classes. The class III enzymes encompass all eukaryotic adenylyl and guanylyl cyclase, and several bacterial and archaebacterial cyclases. Mycobacterial nucleotide cyclases show distinct biochemical properties and domain fusions, and we review here biochemical and structural studies on these enzymes from Mycobacterium tuberculosis and related bacteria. We also present an in silico analysis of nucleotide cyclases found in completely sequenced mycobacterial genomes. It is clear that this group of enzymes demonstrates the tinkering in the class III cyclase domain during evolution, involving subtle structural changes that retain the overall catalytic function and fine tune their activities.     

Sequence diversity of the plasmid replication gene repC in the Rhizobiaceae

The repABC operon is essential for stable maintenance of some Rhizobiaceae plasmids and of pTAV320 from Paracoccus versutus. These plasmids are the largest described family of homologous, yet compatible replicons. The repC gene is essential for plasmid replication, and previous work identified four distinct sequence groups (repC1, repC2, repC3, and repC4) that appear to define different compatibility classes. Probes for these different groups were used to characterize plasmids in Rhizobium leguminosarum population studies and three new repC sequence groups, repC5, repC6, and repC7 were identified. The general repC primers were modified to amplify a wider range of repC sequences and repC sequences were identified in Sinorhizobium and Mesorhizobium type strains. We also showed that the repC3 group-specific primers described previously do not amplify all repC3 sequences and developed a new repC3 amplification strategy.