Secondary Structure Model Research Articles

Cloning and sequence analysis of Galleria mellonella juvenile hormone binding protein--a search for ancestors and relatives.

Juvenile hormone binding proteins (JHBPs) serve as specific carriers of juvenile hormone (JH) in insect hemolymph. As shown in this report, Galleria mellonella JHBP is encoded by a cDNA of 1063 nucleotides. The pre-protein consists of 245 amino acids with a 20 amino acid leader sequence. The concentration of the JHBP mRNA reaches a maximum on the third day of the last larval instar, and decreases five-fold towards pupation. Comparison of amino acid sequences of JHBPs from Bombyx mori, Heliothis virescens, Manduca sexta and G. mellonella shows that 57 positions out of 226 are occupied by identical amino acids. A phylogeny tree was constructed from 32 proteins, which function could be associated to JH. It has three major branches: (i) ligand binding domains of nuclear receptors, (ii) JHBPs and JH esterases (JHEs), and (iii) hypothetical proteins found in Drosophila melanogaster genome. Despite the close positioning of JHEs and JHBPs on the tree, which probably arises from the presence of a common JH binding motif, these proteins are unlikely to belong to the same family. Detailed analysis of the secondary structure modeling shows that JHBPs may contain a beta-barrel motif flanked by alpha-helices and thus be evolutionary related to the same superfamily as calycins.

The European database on small subunit ribosomal RNA.

The European database on SSU rRNA can be consulted via the World WideWeb at http://rrna.uia.ac.be/ssu/ and compiles all complete or nearly complete small subunit ribosomal RNA sequences. Sequences are provided in aligned format. The alignment takes into account the secondary structure information derived by comparative sequence analysis of thousands of sequences. Additional information such as literature references, taxonomy, secondary structure models and nucleotide variability maps, is also available.

The Secondary Structure of Mammalian Mitochondrial 16S rRNA Molecules: Refinements Based on a Comparative Phylogenetic Approach

Eighty six complete 16S ribosomal RNA (rRNA) gene sequences representing every mammalian order (one monotreme, 33 marsupials, 52 placentals) were employed to establish a core secondary structure model for mammalian 16S rRNA. Starting with the Gutell et al. (1993) and De Rijk et al. (1999) models, we used the criteria of potential base-pairing and positional covariance to make refinements in these models for mammalian 16S rRNA molecules. Our results suggest a mammalian secondary structure model with deletions as well as additions to the Gutell et al. (1993) and De Rijk et al. (1999) models for cow. We recognize 53 stems, 41 of which show at least some positional covariance within Mammalia. In addition, we recognize four tertiary interactions. Stems and loops have distinctly different properties, including base composition and relative substitution rates. Accounting for these differences results in improved models of sequence evolution.

Using secondary structure to identify ribosomal numts: cautionary examples from the human genome.

The identification of inadvertently sequenced mitochondrial pseudogenes (numts) is critical to any study employing mitochondrial DNA sequence data. Failure to discriminate numts correctly can confound phylogenetic reconstruction and studies of molecular evolution. This is especially problematic for ribosomal mtDNA genes. Unlike protein-coding loci, whose pseudogenes tend to accumulate diagnostic frameshift or premature stop mutations, functional ribosomal genes are not constrained to maintain a reading frame and can accumulate insertion-deletion events of varying length, particularly in nonpairing regions. Several authors have advocated using structural features of the transcribed rRNA molecule to differentiate functional mitochondrial rRNA genes from their nuclear paralogs. We explored this approach using the mitochondrial 12S rRNA gene and three known 12S numts from the human genome in the context of anthropoid phylogeny and the inferred secondary structure of primate 12S rRNA. Contrary to expectation, each of the three human numts exhibits striking concordance with secondary structure models, with little, if any, indication of their pseudogene status, and would likely escape detection based on structural criteria alone. Furthermore, we show that the unwitting inclusion of a particularly ancient (18-25 Myr old) and surprisingly cryptic human numt in a phylogenetic analysis would yield a well-supported but dramatically incorrect conclusion regarding anthropoid relationships. Though we endorse the use of secondary structure models for inferring positional homology wholeheartedly, we caution against reliance on structural criteria for the discrimination of rRNA numts, given the potential fallibility of this approach.

Identification of antigenic domains on the human sodium-iodide symporter which are recognized by autoantibodies from patients with autoimmune thyroid disease.

The sodium-iodide symporter (NIS) is a novel autoantigen in autoimmune thyroid disease. In the present study we have characterized the antigenic domains on the human symporter which are recognized by autoantibodies from patients with either Graves' disease (GD) or autoimmune hypothyroidism (AH). Deletion derivatives of complementary DNA (cDNA) encoding the Na(+)/I(-) symporter were constructed using polymerase chain reaction (PCR) amplification. These deletion constructs were translated in vitro with the concomitant incorporation of [(35)S]methionine into the protein products. The reactivity of seven GD and six AH sera, which were known to contain symporter-binding antibodies, to each of the radiolabelled modified symporters was then determined in immunoprecipitation experiments. Analyses of the results obtained in the radiobinding assays suggest the existence of multiple antibody binding sites on human NIS (hNIS), including regions between amino acids (aa) 1--134, 191--286, 290--411, 411--520 and 520--588. Computer prediction of the potential B cell epitopes on the symporter revealed that, apart from aa 134--191, all the epitope domains identified overlapped, at least in part, with areas predicted to be highly antigenic. Interestingly, the antigenic domains represented by aa 191--286, 290--411 and 411--520 include regions of the polypeptide which form putative extracellular domains in the secondary structure model of the rat symporter. No correlation between the recognition of specific epitopes on the human symporter and the type of autoimmune thyroid disease was demonstrated.

Distribution of substitution rates and location of insertion sites in the tertiary structure of ribosomal RNA.

The relative substitution rate of each nucleotide site in bacterial small subunit rRNA, large subunit rRNA and 5S rRNA was calculated from sequence alignments for each molecule. Two-dimensional and three-dimensional variability maps of the rRNAs were obtained by plotting the substitution rates on secondary structure models and on the tertiary structure of the rRNAs available from X-ray diffraction results. This showed that the substitution rates are generally low near the centre of the ribosome, where the nucleotides essential for its function are situated, and that they increase towards the surface. An inventory was made of insertions characteristic of the Archaea, Bacteria and Eucarya domains, and for additional insertions present in specific eukaryotic taxa. All these insertions occur at the ribosome surface. The taxon-specific insertions seem to arise randomly in the eukaryotic evolutionary tree, without any phylogenetic relatedness between the taxa possessing them.

Bulinus species on Madagascar: molecular evolution, genetic markers and compatibility with Schistosoma haematobium.

Of the four species of Bulinus found on Madagascar, three species: B. obtusispira, B. liratus and B. bavayi are endemic while the fourth, B. forskalii, is probably a recent introduction from the African mainland. The evolutionary relationships of these species with Bulinus species from Africa were studied by phylogenetic analysis of DNA sequence variation at two mitochondrial loci: cytochrome oxidase subunit I (COI) and large ribosomal subunit (LSU) or 16S. The observed levels of nucleotide divergence within Bulinus were substantial but may underestimate the true levels as there was evidence of 'saturation' of transitional substitutions at both loci. A putative secondary structure model for the sequenced segment of the 16S was developed. Subsequent phylogenetic analysis using transversional changes only for both loci, showed that there were contrasting levels of divergence within the four species groups. B. obtusispira was consistently placed within the B. africanus group, appearing ancestral to this group and was closest to the basal node within Bulinus. Together with B. bavayi, the two species appear to have been isolated on Madagascar for a long time, contrasting with both B. liratus and B. forskalii that appear more recent colonisers; however, estimate of exact times of divergence is problematic. A PCR-RFLP assay was developed to enable identification and discrimination of B. obtusispira and B. liratus using discriminatory variation within the COI. To enable population genetic analysis within B. obtusispira, microsatellite markers were developed using an enrichment method and 8 primer pairs are reported. Laboratory infection experiments using Madasgacan S. haematobium from the Mahabo area showed that certain populations of B. obtusispira, B. liratus and B. bavayi were compatible.

In vitro and in vivo processing of cyanelle tmRNA by RNase P.

Ribonuclease P, the ubiquitous endonuclease required for generating mature tRNA 5' ends, is a ribonucleoprotein in most organisms and organelles, with the exception of mitochondria and chloroplasts of multicellular organisms. The cyanelle of the primitive alga Cyanophora paradoxa is the only photosynthetic organelle where the ribonucleoprotein nature of this enzyme has been functionally proven. tmRNA is another highly structured RNA: it can be aminoacylated with alanine, which is then incorporated into a tag peptide encoded on the same RNA molecule. This dual-function RNA has been found in bacteria, and its gene is also present in mitochondria and plastids from primitive organisms. Since nothing is known about the expression of this RNA in organelles, we have performed processing studies and determined the promoter of cyanelle pre-tmRNA. This RNA is transcribed as a precursor molecule in vivo. Synthetic transcripts of cyanelle pre-tmRNA, including or lacking the mature 3' CCA-end, are efficiently and correctly processed in vitro by bacterial RNase P ribo- and holoenzymes and by the homologous cyanelle RNase P. In addition to these experimental data, we propose a novel secondary structure model for this organellar tmRNA, which renders it more similar to its bacterial counterpart.

Identification of sites in the second exomembrane loop and ninth transmembrane helix of the mammalian Na+/H+ exchanger important for drug recognition and cation translocation.

Mammalian Na(+)/H(+) exchanger (NHE) isoforms are differentially sensitive to inhibition by several distinct classes of pharmacological agents, including amiloride- and benzoyl guanidinium-based derivatives. The determinants of drug sensitivity, however, are only partially understood. Earlier studies of the drug-sensitive NHE1 isoform have shown that residues within the fourth membrane-spanning helix (M4) (Phe(165), Phe(166), Leu(167), and Gly(178)) and a 66-amino acid segment encompassing M9 contribute significantly to drug recognition. In this report, we have identified two residues within M9, one highly conserved (Glu(350)) and the other non-conserved (Gly(356)), that are major determinants of drug sensitivity. In addition, residues in the second exomembrane loop between M3 and M4 (Gly(152), Phe(157), and Pro(158)) were also found to modestly influence drug sensitivity. A double substitution of crucial sites within M4 and M9 of NHE1 with the corresponding residues present in the drug-resistant NHE3 isoform (i.e. L167F/G356A) greatly reduced drug sensitivity in a cooperative manner to levels nearing that of wild type NHE3. The above mutations did not appreciably affect Na(o)(+) affinity but did markedly decrease the catalytic turnover of the transporter. These data suggest that specific sites encompassing M4 and M9 are critical determinants of both drug recognition and cation translocation.

The Balance Beam Function: A New Test Function for the Real World Problems

A new test function called the Balance Beam Function (BBF) is introduced. This function is designed for understanding the performance of evolutionary algorithms on real world problems in which many “neutral networks” are assumed to be found. The concept of a neutral network originates from genotype-phenotype models of RNA secondary structure and protein structure. It refers to long, narrow, meandering, yet flat, pathways through a fitness landscape which may lead to better points in a search space. The objective of the proposed test function is to provide a measure of the speed at which an evolving population moves along a neutral network; this is an important characteristic of an evolutionary algorithm, especially when solving largescale problems. In empirical experiments, two kinds of GA, the standard GA and an extended GA-called the operon-GA-are applied to BBFs in order to investigate their evolutionary behavior and the ease with which their parameters can be tuned.

Evaluation and refinement of tmRNA structure using gene sequences from natural microbial communities.

DNA harvested directly from complex natural microbial communities by PCR has been successfully used to predict RNase P RNA structure, and can potentially provide an abundant source of information for structural predictions of other RNAs. In this study, we utilized genetic variation in natural communities to test and refine the secondary and tertiary structural model for the bacterial tmRNA. The variability of proposed tmRNA secondary structures in different organisms and the lack of any predicted tertiary structure suggested that further refinement of the tmRNA could be useful. To increase the phylogenetic representation of tmRNA sequences, and thereby provide additional data for statistical comparative analysis, we amplified, sequenced, and compared tmRNA sequences from natural microbial communities. Using primers designed from gamma proteobacterial sequences, we determined 44 new tmRNA sequences from a variety of environmental DNA samples. Covariation analyses of these sequences, along with sequences from cultured organisms, confirmed most of the proposed tmRNA model but also provided evidence for a new tertiary interaction. This approach of gathering sequence information from natural microbial communities seems generally applicable in RNA structural analysis.

Evolution and Alignment of the Hypervariable Arm 1 of Aeschynanthus (Gesneriaceae) ITS2 Nuclear Ribosomal DNA

Comparative ITS2 sequencing in the plant genus Aeschynanthus(Gesneriaceae) reveals an insertion/deletion (indel) hot spot in the ITS2 sequences that is difficult to align. Examination of other Gesneriaceae sequences shows that this is a widespread phenomenon in this plant family. Minimum free-energy secondary structure analyses localize the hot spot to the terminal part of arm 1. Arm 1 is twice as long in Gesneriaceae than in other asterids. In addition, the pattern of indels is consistent with this secondary structure model. The high variability of the extended terminal part of arm 1 in Gesneriaceae and the fact that it can be deleted altogether imply that it is functionally superfluous. In contrast, the base of arm 1 is relatively conserved and may function as an exonuclease recognition site. This study illustrates how comparative secondary structure analyses can be helpful in fine-scale alignment. Alignment based on secondary structure conflicts with our initial manual alignment and, to a lesser extent, with a CLUSTAL X alignment with default parameters.

Initiation of HIV-1 Reverse Transcription Is Regulated by a Primer Activation Signal

Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome appears to be strictly regulated at the level of initiation. The primer binding site (PBS), at which the tRNA(3)(Lys) molecule anneals and reverse transcription is initiated, is present in a highly structured region of the untranslated leader RNA. Detailed mutational analysis of the U5 leader stem identified a sequence motif in the U5 region that is critical for activation of the PBS-bound tRNA(3)(Lys) primer. This U5 motif, termed the primer activation signal (PAS), may interact with the TPsiC arm of the tRNA(3)(Lys) primer, similar to the additional interaction proposed for the genome of Rous sarcoma virus and its tRNA(Trp) primer. This suggests that reverse transcription is regulated by a common mechanism in all retroviruses. In HIV-1, the PAS is masked through base pairing in the U5 leader stem. This provides a mechanism for positive and negative regulation of reverse transcription. Based on structure probing of the mutant and wild-type RNAs, an RNA secondary structure model is proposed that juxtaposes the critical PAS and PBS motifs.

The Dimer Initiation Site Hairpin Mediates Dimerization of the Human Immunodeficiency Virus, Type 2 RNA Genome

The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.

Initiation of HIV-2 reverse transcription: a secondary structure model of the RNA-tRNA(Lys3) duplex.

Human immunodeficiency virus type 2 (HIV-2) reverse transcription is initiated from cellular tRNA(Lys3) partially annealed to the RNA viral genome at the primer binding site (PBS). This annealing involves interactions between two highly structured RNA molecules. In contrast to HIV-1, in which the reverse transcription initiation complex has been thoroughly studied, there is still little information regarding a possible model to describe the secondary structure of the template-primer complex in HIV-2. To determine whether HIV-2 RNA sequences flanking the PBS may specifically interact with the natural primer tRNA, we performed site-directed mutagenesis and enzymatic footprinting. An RNA fragment corresponding to the HIV-2 U5 RNA domain and tRNA(Lys3) were probed either in their free form or in the binary complex. Important reactivity changes to nucleases were obtained upon complex formation. In addition to the canonical contacts between the viral PBS and the 3' end acceptor stem of tRNA(Lys3), we identified two additional interacting domains: (i) the U-rich region of the anticodon loop with the A-rich sequence of the internal loop within the U5-prePBS region; (ii) nucleotides 48-54 from the TPsiC domain of tRNA(Lys3) and the 240-247 region of viral U5-RNA. In view of these experimental data and sequence comparison between different HIV-2 isolates, we propose a model for the secondary structure of the HIV-2 template-primer initiation complex.

Derivation of a structural model for the c-myc IRES

We have derived a secondary structure model for the c-myc internal ribosome entry segment (IRES) by using information from chemical probing of the c-myc IRES RNA to constrain structure prediction programs. Our data suggest that the IRES is modular in nature, and can be divided into two structural domains linked by a long unstructured region. Both domains are required for full IRES function. Domain 1 is a complex element that contains a GNNRA apical loop and an overlapping double pseudoknot motif that is topologically unique amongst published RNA structures. Domain 2, the smaller of the two, contains an apical AUUU loop.We have located the ribosome landing site and have shown that ribosomes enter in a 16 nt region downstream of the pseudoknots in a situation similar to that observed in several viral IRESs. To test the structure, several key regions of the IRES were mutated and, interestingly, it appears that some of the structural elements that we have identified function to repress c-myc IRES function. This has profound implications for de-regulation of c-myc expression by mutations occurring in the IRES.

Integrating biocrystallography into traditional biology and chemistry curricula

New academic courses for teaching protein crystallography to biology and chemistry students have been developed. The general aim of the lecture courses is to introduce crystallographic terminology and modern diffraction methodology, to discuss the principles of macromolecular structure, to develop confidence in assessing macromolecular models, and to develop skills in extracting biostructural information from crystallographic literature as well as from bioinformatics resources available on the Internet. Emphasis on structural biology (chemists) or on crystallographic concepts and methodology (biologists) differs depending on the background of the students. In practical classes, the students work with real models of crystals and lattices, and use construction kits to build models of protein secondary structure. Diffraction images of oriented protein crystals are used for determination of unit-cell parameters. An Internet-based multiple-choice quiz is used for tests, self-assessment and practice.

On the origin of the Hirudinea and the demise of the Oligochaeta.

The phylogenetic relationships of the Clitellata were investigated with a data set of published and new complete 18S rRNA gene sequences of 51 species representing 41 families. Sequences were aligned on the basis of a secondary structure model and analysed with maximum parsimony and maximum likelihood. In contrast to the latter method, parsimony did not recover the monophyly of Clitellata. However, a close scrutiny of the data suggested a spurious attraction between some polychaetes and clitellates. As a rule, molecular trees are closely aligned with morphology-based phylogenies. Acanthobdellida and Euhirudinea were reconciled in their traditional Hirudinea clade and were included in the Oligochaeta with the Branchiobdellida via the Lumbriculidae as a possible link between the two assemblages. While the 18S gene yielded a meaningful historical signal for determining relationships within clitellates, the exact position of Hirudinea and Branchiobdellida within oligochaetes remained unresolved. The lack of phylogenetic signal is interpreted as evidence for a rapid radiation of these taxa. The placement of Clitellata within the Polychaeta remained unresolved. The biological reality of polytomies within annelids is suggested and supports the hypothesis of an extremely ancient radiation of polychaetes and emergence of clitellates.

Structural basis for selectivity and toxicity of ribosomal antibiotics.

Ribosomal antibiotics must discriminate between bacterial and eukaryotic ribosomes to various extents. Despite major differences in bacterial and eukaryotic ribosome structure, a single nucleotide or amino acid determines the selectivity of drugs affecting protein synthesis. Analysis of resistance mutations in bacteria allows the prediction of whether cytoplasmic or mitochondrial ribosomes in eukaryotic cells will be sensitive to the drug. This has important implications for drug specificity and toxicity. Together with recent data on the structure of ribosomal subunits these data provide the basis for development of new ribosomal antibiotics by rationale drug design.

Topology of the Na+/dicarboxylate cotransporter: the N-terminus and hydrophilic loop 4 are located intracellularly

The current secondary structure model of the Na+/dicarboxylate cotransporter, NaDC-1, contains 11 transmembrane domains. The model is based on hydropathy analysis and the extracellular location of the carboxy terminus, which contains an N-glycosylation site. In this study, the model was further tested using indirect immunofluorescence of COS-7 cells. The Flag epitope tag (DYKDDDDK) was fused to the amino terminus of NaDC-1 (Flag-NaDC-1), and a monoclonal antibody against the Flag epitope was used to determine the location of the N-terminus. Hydrophilic loop 4 of NaDC-1 was identified using polyclonal antibodies raised against a fusion protein containing amino acids 164–233 of NaDC-1. The expression of NaDC-1 and Flag-NaDC-1 in COS-7 cells was confirmed by functional assays of succinate transport and by Western blots of cell surface biotinylated proteins. Immunofluorescent labeling of cells expressing both NaDC-1 and Flag-NaDC-1 required permeabilization of the plasma membranes with digitonin whereas no immunofluorescence was visible in intact cells. The results of this study show that both the N-terminus and hydrophilic loop 4 of NaDC-1 are located intracellularly, which supports the current model of NaDC-1 structure.