Secondary Structure Model Research Articles

Sequence and conformational preferences at termini of α-helices in membrane proteins: role of the helix environment.

α-Helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α-helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C-termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α-helices in a high-resolution dataset of integral α-helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C-termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near-helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins.

Diverse self-association properties within a family of phage packaging RNAs.

The packaging RNA (pRNA) found in phi29 bacteriophage is an essential component of a molecular motor that packages the phage's DNA genome. The pRNA forms higher-order multimers by intermolecular “kissing” interactions between identical molecules. The phi29 pRNA is a proven building block for nanotechnology and a model to explore the rare phenomenon of naturally occurring RNA self-association. Although the self-association properties of the phi29 pRNA have been extensively studied and this pRNA is used in nanotechnology, the characteristics of phylogenetically related pRNAs with divergent sequences are comparatively underexplored. These diverse pRNAs may lend new insight into both the rules governing RNA self-association and for RNA engineering. Therefore, we used a combination of biochemical and biophysical methods to resolve ambiguities in the proposed secondary structures of pRNAs from M2, GA1, SF5, and B103 phage, and to discover that different naturally occurring pRNAs form multimers of different stoichiometry and thermostability. Indeed, the M2 pRNA formed multimers that were particularly thermostable and may be more useful than phi29 pRNA for many applications. To determine if diverse pRNA behaviors are conferred by different kissing loop sequences, we designed and tested chimeric RNAs based on our revised secondary structural models. We found that although the kissing loops are essential for self-association, the critical determinant of multimer stability and stoichiometry is likely the diverse three-way junctions found in these RNAs. Using known features of RNA three-way junctions and solved structures of phi29 pRNA's junction, we propose a model for how different junctions affect self-association.

Variation in secondary structure of the 16S rRNA molecule in cyanobacteria with implications for phylogenetic analysis.

The analysis of the phylogenetic signal in the sequence of the 16S rRNA gene in cyanobacteria and bacteria has been primarily limited to use of unweighted primary sequence in various cladistic analyses. Such analyses ignore the significance of secondary structure in the 16S rRNA molecule. Structure of this molecule is directly tied to its function, and consequently changes in primary sequence that lead to changes in the secondary structure theoretically have greater evolutionary significance than those changes that do not lead to structural changes. Very little analysis of the impact of primary sequence data on the secondary structure of the small subunit rRNA molecule in cyanobacteria has been reported. Herein is reported a secondary structure model of the full 16S rRNA sequence for Nostoc commune Vaucher ex Bornet et Flahault. We also assess the variability in secondary structure of six variable helices in 180 OTUs of cyanobacteria. Variations were minor in most instances, and the observed motifs were not only similar among these cyanobacteria but also were similar to Escherichia coli, a well-studied, representative non-photosynthetic bacterium. Helix 17 was the most variable helix in terms of structure and length of primary sequence. In order to maintain the motifs, we assumed the presence of non-canonical base pairings in the helices. Phylogenies using primary sequence data alone and using primary sequence data in conjunction with coding for secondary structure were very similar, although secondary structural considerations produced trees that more closely reflected the current ultrastructure-based modern taxonomy in the group.

Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme.

Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5' to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5' to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2'-hydroxyl of the nucleotide 5' to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket.

Virtual screening of gene expression regulatory sites in non-coding regions of the infectious salmon anemia virus

BackgroundMembers of the Orthomyxoviridae family, which contains an important fish pathogen called the infectious salmon anemia virus (ISAV), have a genome consisting of eight segments of single-stranded RNA that encode different viral proteins. Each of these segments is flanked by non-coding regions (NCRs). In other Orthomyxoviruses, sequences have been shown within these NCRs that regulate gene expression and virulence; however, only the sequences of these regions are known in ISAV, and a biological role has not yet been attributed to these regions. This study aims to determine possible functions of the NCRs of ISAV.ResultsThe results suggested an association between the molecular architecture of NCR regions and their role in the viral life cycle. The available NCR sequences from ISAV isolates were compiled, alignments were performed to obtain a consensus sequence, and conserved regions were identified in this consensus sequence. To determine the molecular structure adopted by these NCRs, various bioinformatics tools, including RNAfold, RNAstructure, Sfold, and Mfold, were used. This hypothetical structure, together with a comparison with influenza, yielded reliable secondary structure models that lead to the identification of conserved nucleotide positions on an intergenus level. These models determined which nucleotide positions are involved in the recognition of the vRNA/cRNA by RNA-dependent RNA polymerase (RdRp) or mRNA by the ribosome.ConclusionsThe information obtained in this work allowed the proposal of previously unknown sites that are involved in the regulation of different stages of the viral cycle, leading to the identification of new viral targets that may assist future antiviral strategies.

Challenges of Public Health Nurses in Coordinating Relationships: Scale Development

We developed a scale for measuring the challenges faced by public health nurses in coordinating relationships for supporting preschool children with autism spectrum disorder, and examined the scale's construct validity using a factorial structure model. Participants were 708 Japanese public health nurses. The secondary structural model consisted of 6 factors with 25 items. Internal consistency and reliability were high and confirmatory factor analysis using structural equation modeling indicated that the fit criteria were statistically significant. Attributes of public health nurses were significantly related to scale scores. These findings validate the efficacy of our scale to identify and assess the challenges of public health nurses in relationship coordination.

RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP).

Many biological processes are RNA-mediated, but higher-order structures for most RNAs are unknown, making it difficult to understand how RNA structure governs function. Here we describe SHAPE mutational profiling (SHAPE-MaP) that makes possible de novo and large-scale identification of RNA functional motifs. Sites of 2’-hydroxyl acylation by SHAPE are encoded as non-complementary nucleotides during cDNA synthesis, as measured by massively parallel sequencing. SHAPE-MaP-guided modeling identified greater than 90% of accepted base pairs in complex RNAs of known structure and was used to define a second-generation model for the HIV-1 RNA genome. The HIV-1 model contains all known structured motifs and previously unknown elements, including experimentally validated pseudoknots. SHAPE-MaP yields accurate and high-resolution secondary structure models, enables analysis of low abundance RNAs, disentangles sequence polymorphisms in single experiments, and will ultimately democratize RNA structure analysis.

A tractable genotype–phenotype map modelling the self-assembly of protein quaternary structure

The mapping between biological genotypes and phenotypes is central to the study of biological evolution. Here, we introduce a rich, intuitive and biologically realistic genotype–phenotype (GP) map that serves as a model of self-assembling biological structures, such as protein complexes, and remains computationally and analytically tractable. Our GP map arises naturally from the self-assembly of polyomino structures on a two-dimensional lattice and exhibits a number of properties: redundancy (genotypes vastly outnumber phenotypes), phenotype bias (genotypic redundancy varies greatly between phenotypes), genotype component disconnectivity (phenotypes consist of disconnected mutational networks) and shape space covering (most phenotypes can be reached in a small number of mutations). We also show that the mutational robustness of phenotypes scales very roughly logarithmically with phenotype redundancy and is positively correlated with phenotypic evolvability. Although our GP map describes the assembly of disconnected objects, it shares many properties with other popular GP maps for connected units, such as models for RNA secondary structure or the hydrophobic-polar (HP) lattice model for protein tertiary structure. The remarkable fact that these important properties similarly emerge from such different models suggests the possibility that universal features underlie a much wider class of biologically realistic GP maps.

Genomic characterization of a novel picornavirus in Pekin ducks

A novel picornavirus was detected from Pekin ducks (Anas platyrhynchos domestica) and completely sequenced. The virus was most closely related to megriviruses, with amino acid identities of 32–68%, 35–45%, 51–57%, 41–50%, and 61–63% in the P1, P2, P3, polyprotein, and 2C and 3CD regions, respectively. The virus was thus identified as an additional species in the genus Megrivirus and named Duck megrivirus (DMV). Sequence analyses indicated that the DMV genome possessed a megrivirus-like organization and also exhibited several unique features. The polyadenylated genome comprised 9700nt, one of the largest among known picornaviruses. A notable feature was the 2A region, which had an association of two distinct, function-unknown 2As (2A1 and 2A2) and a parechovirus-like 2A3. The 5′ untranslated region (UTR) contained a variant type IVB internal ribosome entry site (IRES), which possessed a long helix III4 ending with the “8”-like 20-nt-long conserved structure at the top of domain III. The secondary structure model of inferred domain III of DMV-like IRES was also conserved in quail picornavirus, pigeon picornavirus B, and megriviruses. Domain II in DMV contained the conserved internal and apical loops previously identified in groups A and C of hepacivirus/pestivirus like IRESs. Moreover, DMV was closely related to different megriviruses in different genomic regions. These findings suggest that recombination events involving exchange of coding and noncoding regions may have occurred. DMV was detected in 28 of 117 (23.9%) ducks from four provinces in China, suggesting a high prevalence of DMV in duck populations.

RNA secondary structure modeling at consistent high accuracy using differential SHAPE.

RNA secondary structure modeling is a challenging problem, and recent successes have raised the standards for accuracy, consistency, and tractability. Large increases in accuracy have been achieved by including data on reactivity toward chemical probes: Incorporation of 1M7 SHAPE reactivity data into an mfold-class algorithm results in median accuracies for base pair prediction that exceed 90%. However, a few RNA structures are modeled with significantly lower accuracy. Here, we show that incorporating differential reactivities from the NMIA and 1M6 reagents--which detect noncanonical and tertiary interactions--into prediction algorithms results in highly accurate secondary structure models for RNAs that were previously shown to be difficult to model. For these RNAs, 93% of accepted canonical base pairs were recovered in SHAPE-directed models. Discrepancies between accepted and modeled structures were small and appear to reflect genuine structural differences. Three-reagent SHAPE-directed modeling scales concisely to structurally complex RNAs to resolve the in-solution secondary structure analysis problem for many classes of RNA.

The complete mitochondrial genome of Cervaphis quercus (Insecta: Hemiptera: Aphididae: Greenideinae)

The mitochondrial genome of Cervaphis quercus has been sequenced and annotated. The entire genome of 15,272 bp encodes two ribosomal RNA genes (rrnL and rrnS), 22 transfer RNA (tRNA) genes, 13 protein-coding genes and a control region. The genome has the same gene order as that found in the inferred ancestral insect. Nucleotide composition is highly A+T biased. All protein-coding genes use standard mitochondrial initiation codons. Secondary structure models of the two ribosomal RNA genes of C. quercus are similar to those proposed for other insects. All tRNAs have the classic clover-leaf structure, except for the dihydrouridine (DHU) arm of trnS (AGN), which forms a simple loop. The presence of structural elements in the control region is also discussed, with an emphasis on the possible regulation of replication and/or transcription. Comparison with mitochondrial genomes of other aphid species shows their gene arrangements are conserved; however, the variety of repeat regions in species from a different aphid subfamily, Aphidinae, suggests that they resulted from independent evolutionary events.

An immature retroviral RNA genome resembles a kinetically trapped intermediate state.

Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. The structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.

An Unidentified Parasitoid Community (Chalcidoidea) is Associated with Pine-FeedingChionaspisScale Insects (Hemiptera: Diaspididae)

AbstractMany of the >22,000 described species of parasitoids in the Chalcidoidea attack armored scale insects, a group that may contain many cryptic species. Because the diversity of both these groups may be high, associations between them are likely to be underreported, and accurate reporting requires direct association between parasitoids and their scale insect hosts. One group of well-known armored scale insects are the pine-feeding Chionaspis of North America, which have been considered to be two species of economic pests, and have over a century of parasitoid records. These Chionaspis have been revealed to be a complex of at least 10, mostly cryptic, species. We have sequenced chalcidoid 28S rDNA from 50 parasitized Chionaspis hosts. We explore these host-parasitoid associations by placing the parasitoid sequences in a phylogenetic context. We aligned the sequences manually, based on secondary structure models, and added them to a previous 28S secondary structure alignment for the Chalcidoidea that includes 19 families, 72 subfamilies, 369 genera, and 685 species. The results of maximum likelihood analyses were interpreted using an updated, comprehensive list of parasitoids recorded from pine-feeding Chionaspis. The sequences are placed in nine clades, only two of which appear consistent with known Chionaspis parasitoids. Although pine-feeding Chionaspis are among the best-known armored scales, our results indicate that their parasitoid diversity has been underreported.

Kinetics of DNA duplex formation: A-tracts versus AT-tracts.

The hybridisation and melting of DNA strands are critical steps in many biological processes, but still a deeper understanding of the kinetics is lacking. This is evident from the absence of a clear correlation between rate constants for duplex formation and the number of bases in the strand or the sequence. Here we have probed differences between formation times of A-tracts and AT-tracts by studying complementary model strands mainly comprised of adenine (A) and thymine (T) in stopped-flow (SF) experiments. These strands are relevant as DNA replication begins in regions with a large number of AT base pairs. Interpretation of our results is aided by secondary-structure modelling where both the fractions of the different types of structures and the number of paired bases in the lowest-energy ones are determined. The model is based on calculation of free energies using fixed values for enthalpies and entropies associated with base pairing and a stochastic sampling of the possible structures. We find that the strand length affects rates: the activation energy for the formation of short (16-base pairs) A-tracts is larger than that for longer ones (20-base pairs). Activation energies for the formation of AT-tracts are an order of magnitude larger, and larger for shorter strands than for long ones. These higher activation energies are in agreement with the fact that the fraction of unpaired bases in the constituent AT-tract strands is less than in those which comprise the A-tracts. That the pre-structures of the single strands significantly affect rates is also used to rationalise the results obtained for two pairs of complementary 12-mer strands that have the same bases but in a different sequence; we report here similar activation energies as reported earlier and that these are strongly sequence dependent. Finally, we demonstrate that SF can be coupled with the measurement of circular dichroism (CD) in the vacuum ultraviolet (VUV) region, taking advantage of a synchrotron radiation facility, and that CD is useful to probe geometrical structures in the VUV where the absorption by DNA is high. Though this work is preliminary, our initial results suggest that the strands align prior to the formation of base pairs.

SHAPE analysis of small RNAs and riboswitches.

We describe structural analysis of small RNAs by SHAPE chemical probing. RNAs are treated with 1-methyl-7-nitroisatoic anhydride, a reagent that detects local nucleotide flexibility; and N-methylisatoic anhydride and 1-methyl-6-nitroisatoic anhydride, reagents which together detect higher-order and noncanonical interactions. Chemical adducts are quantified as stops during reverse transcriptase-mediated primer extension. Probing information can be used to infer conformational changes and ligand binding and to develop highly accurate models of RNA secondary structures.

DREB1A from Oryza sativa var. IR6: homology modelling and molecular docking

The family of AP2/ERF plant specific transcription factors, including dehydration-responsive element binding (DREB) proteins, has been reported to activate and bind with the dehydration responsive element/C repeat (DRE/CRT) of stress-inducible gene promoters. We amplified DREB1A from Oryza sativa var. IR6 (807 bp) and subjected it to protein homology modelling and phylogenetic analysis. BLAST sequence analysis indicated high similarity (99%) with the O. sativa japonica group. Phylogenetic analysis was carried out with other 12 DREB sequences from cereals/grasses using Clustal Omega. Protein secondary structure and 3D models were determined using SOPMA and MODELLER v9.10, respectively. PROSA was used to determine the excellence and consistency of models. The results indicated that the Z-score value was -2.94 and 79.5% of the residues were found in the favoured region. A psi/phi Ramachandran plot was determined using PROCHECK. The molecular docking of the GCC-box binding domain (GBD) of the protein under investigation was done using atomic coordinates of Arabidopsis thaliana GBD- and GCC-box containing DNA, respectively. The tertiary structure of the modelled protein and template were found to be very similar as predicted by a root mean square deviation of 0.968 A. It seemed that both the proteins interacted with the major groove of DNA using Beta-sheets. We attempted to expose the molecular basis of O. sativa var. IR6 DREB1A protein interaction with the target promoter sequence. The results highlighted that this gene could be a good candidate for production of abiotic stress-tolerant crop(s) in the future.

RNA secondary structure modeling at consistent high accuracy using differential SHAPE

RNA secondary structure modeling at consistent high accuracy using differential SHAPE

Conformational analysis of the full‐length M2 protein of the influenza A virus using solid‐state NMR

The influenza A M2 protein forms a proton channel for virus infection and mediates virus assembly and budding. While extensive structural information is known about the transmembrane helix and an adjacent amphipathic helix, the conformation of the N-terminal ectodomain and the C-terminal cytoplasmic tail remains largely unknown. Using two-dimensional (2D) magic-angle-spinning solid-state NMR, we have investigated the secondary structure and dynamics of full-length M2 (M2FL) and found them to depend on the membrane composition. In 2D (13)C DARR correlation spectra, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-bound M2FL exhibits several peaks at β-sheet chemical shifts, which result from water-exposed extramembrane residues. In contrast, M2FL bound to cholesterol-containing membranes gives predominantly α-helical chemical shifts. Two-dimensional J-INADEQUATE spectra and variable-temperature (13)C spectra indicate that DMPC-bound M2FL is highly dynamic while the cholesterol-containing membranes significantly immobilize the protein at physiological temperature. Chemical-shift prediction for various secondary-structure models suggests that the β-strand is located at the N-terminus of the DMPC-bound protein, while the cytoplasmic domain is unstructured. This prediction is confirmed by the 2D DARR spectrum of the ectodomain-truncated M2(21-97), which no longer exhibits β-sheet chemical shifts in the DMPC-bound state. We propose that the M2 conformational change results from the influence of cholesterol, and the increased helicity of M2FL in cholesterol-rich membranes may be relevant for M2 interaction with the matrix protein M1 during virus assembly and budding. The successful determination of the β-strand location suggests that chemical-shift prediction is a promising approach for obtaining structural information of disordered proteins before resonance assignment.

The mitochondrial genome of the plant bug Apolygus lucorum (Hemiptera: Miridae): Presently known as the smallest in Heteroptera

The complete mitochondrial (mt) genome of the plant bug, Apolygus lucorum, an important cotton pest, has been sequenced and annotated in this study. The entire circular genome is 14 768 bp in size and represents the smallest in presently known heteropteran mt genomes. The mt genome is encoding for two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, 13 protein coding genes and a control region, and the order, content, codon usage and base organization show similarity to a great extent to the hypothetical ancestral model. All protein coding genes use standard initiation codons ATN. Conventional stop codons TAA and TAG have been assigned to the most protein coding genes; however, COIII, ND4 and ND5 genes show incomplete terminator signal (T). All tRNA genes possess the typical clover leaf structure, but the dihydrouridine arm of tRNA(Ser(AGN)) only forms a simple loop. Secondary structure models of rRNA genes are generally in accordance with the former models, although some differences exist in certain parts. Three intergenic spacers have never been found in sequenced mt genomes of Heteroptera. The phylogenetic study based on protein coding genes is largely congruent with previous phylogenetic work. Both Bayesian inference and maximum likelihood analyses highly support the sister relationship of A. lucorum and Lygus lineolaris, and Miridae presents a sister position to Anthocoridae.

Association between a Tetranucleotide Repeat Polymorphism of SPAG16 Gene and Cataract in Male Children

Purpose. Studies involving genotyping of STR markers at 2q34 have repeatedly found the region to host the disease haplotype for pediatric cataract. Present study investigated the association of D2S2944 marker, in sperm associated antigen 16 (SPAG16) gene and rs2289917 polymorphism, in γ-crystallin B gene, with childhood cataract. Methods. 97 pediatric cataract cases and 110 children with no ocular defects were examined for tetranucleotide repeat marker/SNP using PCR-SSLP/RFLP techniques. Polymorphisms were assessed for association using contingency tables and linkage disequilibrium among alleles of the markers was estimated. Energy-optimization program predicted the secondary structure models of repeats of D2S2944. Results. Seven alleles of D2S2944, with 9–15 “GATA” repeats, were observed. Frequency of the longer allele of D2S2944, ≥(GATA)13 repeats, was 0.73 in cases and 0.56 in controls (P = 0.0123). Male children bearing ≥(GATA)13 repeats showed >3-fold higher risk for cataract (CI95% = 1.43–7.00, P = 0.0043, P c = 0.0086) as compared to female children (OR = 1.19, CI95% = 0.49–2.92, P = 0.70). Cases with haplotype—≥(GATA)13 of D2S2944 and “C” allele rs2289917—have a higher risk for pediatric cataract (OR = 2.952, CI95% = 1.595~5.463, P = 0.000453). >(GATA)13 repeats formed energetically more favorable stem-loop structure. Conclusion. Intragenic microsatellite repeat expansion in SPAG16 gene increases predisposition to pediatric cataract by probably interfering posttranscriptional events and affecting the expression of adjacent lens transparency gene/s in a gender bias manner.