Secondary Structure Model Research Articles

Evaluation of the Dynamic Structure of DsrA RNA from E. coli and Its Functional Consequences

DsrA RNA is an 87-nucleotide regulatory non-protein-coding RNA of Escherichia coli for which two secondary structure models (I and II) have been proposed. We have compared these models by the energy calculations, which revealed that the currently accepted model II should be rejected on the basis of thermodynamics. Here we provide new results of nuclease footprinting analysis and the application of RNA technologies that have not previously been used for DsrA RNA structural studies, such as hydrolysis with RNase H, DNAzyme, hydroxyl radicals and lead. These approaches together with bioinformatics calculations provided strong arguments for a new model III. This model clearly shows that the long U-rich region between hairpins 1 and 2 is double-stranded. These findings shed new light on DsrA RNA-Hfq interactions.

Characteristics of the glmS ribozyme suggest only structural roles for divalent metal ions

The glmS ribozyme is a riboswitch class that occurs in certain Gram-positive bacteria, where it resides within mRNAs encoding glucosamine 6-phosphate synthase. Members of this self-cleaving ribozyme class rapidly catalyze RNA transesterification upon binding GlcN6P, and genetic evidence suggests that this cleavage event is important for down-regulating GlmS protein expression. In this report, we present a refined secondary structure model of the glmS ribozyme and determine the importance of a conserved pseudoknot structure for optimal ribozyme function. Analyses of deletion constructs demonstrate that the pseudoknot, together with other structural elements, permits the ribozyme to achieve maximum rate constants for RNA cleavage at physiologically relevant Mg2+ concentrations. In addition, we show that substantial rate enhancements are supported by an exchange-inert cobalt (III) complex and by molar concentrations of monovalent ions. Our findings indicate that the glmS ribozyme forms a complex structure to employ catalytic strategies that do not require the direct participation of divalent metal ions.

Analysis of the Secondary Structure of Expansion Segment 39 in Ribosomes from Fungi, Plants and Mammals

The structure of expansion segment 39, ES39, in eukaryotic 23 S-like ribosomal RNA was analysed using a combination of chemical and enzymic reagents. Ribosomes were isolated from yeast, wheat, mouse, rat and rabbit, five organisms representing three different eukaryotic kingdoms. The isolated ribosomes were treated with structure-sensitive chemical and enzymic reagents and the modification patterns analysed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The expansion segment was relatively accessible to modification by both enzymic and chemical probes, suggesting that ES39 was exposed on the surface of the ribosomes. The collected modification data were used in secondary structure modelling of the expansion segment. Despite considerable variation in both sequence and length between organisms from different kingdoms, the structure analysis of the expansion segment gave rise to structural fingerprints that allowed identification of homologous structures in ES39 from fungi, plants and mammals. The homologous structures formed an initial helix and an invariant hairpin connected to the initial helix via a long single-stranded loop. The remaining part of the ES39 sequences accounted for most of the length variation seen between the analysed species. This part could form additional, albeit less similar, hairpins. A comparison of ES39 sequences from other fungi, plants and mammals showed that identical structures could be formed in these organisms.

Functional Analyses of RNA Structures Shared between the Internal Ribosome Entry Sites of Hepatitis C Virus and the Picornavirus Porcine Teschovirus 1 Talfan

The internal ribosome entry site (IRES) of porcine teschovirus 1 (PTV-1), a member of the Picornaviridae family, is quite distinct from other well-characterized picornavirus IRES elements, but it displays functional similarities to the IRES from hepatitis C virus (HCV), a member of the Flaviviridae family. In particular, a dominant negative mutant form of eIF4A does not inhibit the activity of the PTV-1 IRES. Furthermore, there is a high level (ca. 50%) of identity between the PTV-1 and HCV IRES sequences. A secondary-structure model of the whole PTV-1 IRES has been derived which includes a pseudoknot. Validation of specific features within the model has been achieved by mutagenesis and functional assays. The differences and similarities between the PTV-1 and HCV IRES elements should assist in defining the critical features of this type of IRES.

Cloning and characterization of a novel trypsin inhibitor (BTIw1) gene fromFagopyrum esculentum

Based on the amino acid information of trypsin inhibitor of buckwheat (Fagopyrum Esculentum Moench), degenerated primers were designed and a full-length cDNA sequence named BTIomega1 (Buckwheat Trypsin Inhibitor) was amplified from the leaves RNA by using RT-PCR and rapid amplification of cDNA ends (RACE) methods. Sequence analysis shows that the 392 bp cDNA contained an open reading frame (ORF) of 216 bp, encoding 72 amino acids residues. The deduced amino acid sequence exhibits 96 and 93% homology with BWI-1 and BTI-2, a natural trypsin inhibitor from buckwheat seeds. Southern blotting suggested that three copies of BTIomega1 gene existed in the buckwheat genome. Moreover, a predicted secondary structure and 3D-structural model was constructed by homology modeling. To our knowledge, this is the first all-round report of the gene BTIomega1. The novel BTIomega1 gene has been submitted to the GeneBank under Accession No. DQ289792.

Towards more realistic dynamical models for DNA secondary structure

We propose a dynamical model for the secondary structure of DNA, which is based on the finite stacking enthalpies used in thermodynamics calculations. In this model, the two strands can separate and the bases are allowed to rotate perpendicular to the sequence axis. We show, through molecular dynamics simulations, that the model has the correct behaviour at the denaturation transition.

A molecular phylogenetic study of armoured scale insects (Hemiptera: Diaspididae)

Abstract. Armoured scale insects are economically important parasites of woody plants and grasses. They are promising subjects for the evolutionary study of physiology (no complete gut), genetics (chimerism, paternal genome elimination, frequent parthenogenesis) and coevolution (with host plants, parasitoids, Septobasidium fungi, endosymbiotic bacteria). Little phylogenetic work has been accomplished with armoured scales, and uncertainty surrounds their classification. Here, we report the phylogenetic results of Bayesian and parsimony analyses of 705 base pairs of Elongation Factor 1α and 660 base pairs of 28S from eighty‐nine species of armoured scale insects, representing forty‐seven genera and five tribes in the subfamilies Diaspidinae and Aspidiotinae, together with two outgroups. 28S was aligned based on a secondary structural model. Our results broadly corroborate the major features of the existing classification, although we do not find perfect monophyly of any of the traditionally recognized subfamilies or tribes. The subfamily Aspidiotinae is paraphyletic with respect to the subfamily Diaspidinae. Diaspidinae consists of two main clades that only roughly correspond to the tribes Lepidosaphidini and Diaspidini. Diaspidini is nearly monophyletic, except that it includes a single aspidiotine species. Other members of the tribe Aspidiotini form a clade, except that the clade includes a single species of Leucaspidini and excludes Maskellia and Pseudaonidia. Our results weakly support the hypothesis that the most recent common ancestor of the Diaspididae had adult females that were permanently enclosed within the derm of the second instar (the pupillarial habit) and had diploid adult males that eliminated their paternal genomes during spermatogenesis (late paternal genome elimination).

Effect of Mutations in the Mouse Hepatitis Virus 3′(+)42 Protein Binding Element on RNA Replication

The mouse hepatitis virus (MHV) genome's 3' untranslated region contains cis-acting sequences necessary for replication. Studies of MHV and other coronaviruses have indicated a role for RNA secondary and tertiary elements in replication. Previous work in our laboratory has identified four proteins which form ribonucleoprotein complexes with the 3'-terminal 42 nucleotides [3'(+)42] of the MHV genome. Defective interfering (DI) RNA replication assays have demonstrated a role for the 3'(+)42 host protein binding element in the MHV life cycle. Using gel mobility shift RNase T1 protection assays and secondary structure modeling, we have characterized a possible role for RNA secondary structure in host protein binding to the 3'-terminal 42-nucleotide element. Additionally we have identified a role for the 3'-terminal 42-nucleotide host protein binding element in RNA replication and transcription using DI RNA replication assays and targeted recombination and by directly constructing mutants in this protein binding element using a recently described MHV reverse genetic system. DI RNA replication assays demonstrated that mutations in the 3'(+)42 host protein binding element had a deleterious effect on the accumulation of DI RNA. When the identical mutations were directly inserted into the MHV genome, most mutant genomes were viable but formed smaller plaques than the wild-type parent virus. One mutant was not viable. This mutant directed the synthesis of genome-sized negative-sense RNA approximately as efficiently as the wild type did but had a defect in subgenomic mRNA synthesis. These results point to a potential role for sequences at the extreme 3' end of the MHV genome in subgenomic RNA synthesis.

Identification of evolutionary conserved structural elements in the mt SSU rRNA of Zygaenoidea (Lepidoptera): A comparative sequence analysis

Knowledge of the secondary structure of ribosomal RNA (rRNA) molecules has become increasingly important in phylogenetic analyses. Advances in RNA substitution models have underlined the need for reliable secondary-structure models for individual taxonomic groups. The present investigation aims to infer a secondary-structure model of the mt SSU (12S) rRNA of Zygaenoidea using a comparative approach. Structural variation of the 12S rRNA molecule proves to be minor among the investigated species, although at least two helices exhibit taxon-specific deviations. The consensus structure of the zygaenoid mt SSU rRNA clearly differs from the structure published for Bombyx mori and challenges some helices proposed in the silk moth model. Our analyses demonstrate the need for taxon-specific rRNA models, which can capture evolutionary patterns in these molecules far better than general eukaryotic consensus structures and thus provide an improved basis for phylogenetic analyses incorporating secondary-structure information.

A Hexapod nuclear SSU rRNA secondary‐structure model and catalog of taxon‐specific structural variation

RNA molecules and in particular the nuclear SSU RNA play an important role in molecular systematics. With the advent of increasingly parameterized substitution models in systematic research, the incorporation of secondary-structure information became a realistic option compensating interdependence of character variation. As a prerequisite, consensus structures of eukaryotic SSU RNA molecules have become available through extensive comparative analyses and crystallographic studies. Despite extensive research in hexapod phylogenetics, consensus SSU RNA secondary structures focusing on hexapods have not yet been explored. In this study, we compiled a representative hexapod SSU data set of 261 sequences and inferred a specific consensus SSU secondary-structure model. Our search for conserved structural motives relied on a combined approach of thermodynamic and covariation analyses. The hexapod consensus-structure model deviates from the canonical eukaryotic model in a number of helices. Additionally, in several helices the hexapod sequences did not support a single consensus structure. We provide consensus structures of these sections of single less-inclusive taxa, thus facilitating the adaptation of the consensus hexapod model to less-inclusive phylogenetic questions. The secondary-structure catalog will foster the application of RNA structure models in phylogenetic analyses using the SSU rRNA molecule, and it will improve the realism of substitution models and the reliability of reconstructions based on rRNA sequences.

Phylogenetic relationships and species differentiation of 39 Legionella species by sequence determination of the RNase P RNA gene rnpB

The rnpB gene is ubiquitous in Bacteria, Archaea and Eucarya and encodes the RNA component of RNase P, an endoribonuclease P that consists of one RNA and one protein subunit (C5). In this study, partial rnpB genes were sequenced from 39 type strains and 16 additional strains of the genus Legionella. Models of the putative secondary structures of the RNase P RNA in the genus Legionella are proposed and possible interactions between RNase P RNA and C5 are discussed. The phylogenetic relationships within the genus Legionella were examined and rnpB sequences indicated six main clades that together comprised 27 of the 39 species examined. The phylogenetic relationships were further inferred by analysing combined datasets of sequences from the rnpB, mip, 16S rRNA and rpoB genes. It is concluded that rnpB is suitable for use in phylogenetic studies of closely related species and that it exhibits the potential to discriminate between Legionella species.

The U11/U12 snRNP 65K protein acts as a molecular bridge, binding the U12 snRNA and U11-59K protein

U11 and U12 interact cooperatively with the 5' splice site and branch site of pre-mRNA as a stable preformed di-snRNP complex, thereby bridging the 5' and 3' ends of the intron within the U12-dependent prespliceosome. To identify proteins contributing to di-snRNP formation and intron bridging, we investigated protein-protein and protein-RNA interactions between components of the U11/U12 snRNP. We demonstrate that the U11/U12-65K protein possesses dual binding activity, interacting directly with U12 snRNA via its C-terminal RRM and the U11-associated 59K protein via its N-terminal half. We provide evidence that, in contrast to the previously published U12 snRNA secondary structure model, the 3' half of U12 forms an extended stem-loop with a highly conserved seven-nucleotide loop and that the latter serves as the 65K binding site. Addition of an oligonucleotide comprising the 65K binding site to an in vitro splicing reaction inhibited U12-dependent, but not U2-dependent, pre-mRNA splicing. Taken together, these data suggest that U11/U12-65K and U11-59K contribute to di-snRNP formation and intron bridging in the minor prespliceosome.

Identification and analysis of ribonuclease P and MRP RNA in a broad range of eukaryotes

RNases P and MRP are ribonucleoprotein complexes involved in tRNA and rRNA processing, respectively. The RNA subunits of these two enzymes are structurally related to each other and play an essential role in the enzymatic reaction. Both of the RNAs have a highly conserved helical region, P4, which is important in the catalytic reaction. We have used a bioinformatics approach based on conserved elements to computationally analyze available genomic sequences of eukaryotic organisms and have identified a large number of novel nuclear RNase P and MRP RNA genes. For MRP RNA for instance, this investigation increases the number of known sequences by a factor of three. We present secondary structure models of many of the predicted RNAs. Although all sequences are able to fold into the consensus secondary structure of P and MRP RNAs, a striking variation in size is observed, ranging from a Nosema locustae MRP RNA of 160 nt to much larger RNAs, e.g. a Plasmodium knowlesi P RNA of 696 nt. The P and MRP RNA genes appear in tandem in some protists, further emphasizing the close evolutionary relationship of these RNAs.

MOLECULAR PHYLOGENY OF STAURASTRUM MEYEN EX RALFS AND RELATED GENERA (ZYGNEMATOPHYCEAE, STREPTOPHYTA) BASED ON CODING AND NONCODING RDNA SEQUENCE COMPARISONS1

Nuclear‐encoded small subunit rDNA, 1506 group I intron, and internal transcribed spacer sequences were obtained from 39 strains representing five core desmid genera, Staurastrum, Staurodesmus Teil., Cosmarium Corda ex Ralfs, Xanthidium Ehr. ex Ralfs, and Euastrum Ehr. ex Ralfs (Desmidiaceae, Zygnematophyceae), and used individually and concatenated to assess phylogenetic relationships between putatively allied members of the family. To identify positional homology between divergent noncoding sequences, secondary structure models were generated and their reliability assessed by screening the alignment for compensating base changes. The phylogeny based on coding and noncoding sequence comparisons confidently resolved a monophyletic core of the genus Staurastrum but also revealed the artificial nature of the traditional genus. Twenty distinct species representing a wide range of morphotypes of Staurastrum formed a strongly supported generic clade that was further split into three well‐resolved lineages. The phylogenetic relationships revealed within Staurastrum were in conflict with all previous formal or informal classifications of the genus. The genera Staurodesmus and Cosmarium were shown to be highly polyphyletic, and some morphologically similar taxa displayed high sequence divergence that exceeded generic boundaries. Apparently, the taxonomic significance of some morphological characters in Staurastrum and other desmid genera has been greatly overestimated.

Secondary structure probing of the human RNase MRP RNA reveals the potential for MRP RNA subsets

RNase MRP is a ribonucleoprotein endoribonuclease involved in eukaryotic pre-rRNA processing. The enzyme possesses an RNA subunit, structurally related to that of RNase P RNA, that is thought to be catalytic. RNase MRP RNA sequences from Saccharomycetaceae species are structurally well defined through detailed phylogenetic and structural analysis. In contrast, higher eukaryote MRP RNA structure models are based on comparative sequence analysis of only five sequences and limited probing data. Detailed structural analysis of the Homo sapiens MRP RNA, entailing enzymatic and chemical probing, is reported. The data are consistent with the phylogenetic secondary structure model and demonstrate unequivocally that higher eukaryote MRP RNA structure differs significantly from that reported for Saccharomycetaceae species. Neither model can account for all of the known MRP RNAs and we thus propose the evolution of at least two subsets of RNase MRP secondary structure, differing predominantly in the predicted specificity domain.

Lead(II) cleavage analysis of RNase P RNA in vivo

The overall conformation of M1 RNA, the catalytic RNA subunit of RNase P in Escherichia coli, was analyzed in vivo and, in the presence of the C5 protein subunit, in vitro by lead(II) acetate probing. The partial cleavage patterns obtained are congruent with previous structure mapping performed in vitro. Most of the known major and minor cleavages in M1 RNA were supported and could be mapped onto a secondary structure model. The data obtained indicate that C5 has only minor effects on the overall structure of the RNA subunit. The similar cleavage patterns obtained in vitro and in vivo furthermore suggest that the intracellular environment does not greatly alter the overall conformation of M1 RNA within the holoenzyme complex. Moreover, our data indicate that M1 RNA in vivo is present in at least two states-the major fraction is bound to tRNA substrates and a minor fraction is substrate free. Finally, both in this and previous work we found that lead(II) probing data from in vivo experiments conducted on longer RNAs (tmRNA and M1 RNA) generally gives superior resolution compared to parallel in vitro experiments. This may reflect the absence of alternative conformers present in vitro and the more natural state of these RNAs in the cell due to proper, co-transcriptional folding pathways and possibly the presence of RNA chaperones.

Aligned 18S for Zoraptera (Insecta): Phylogenetic position and molecular evolution

The order Zoraptera (angel insects) is one of the least known insect groups, containing only 32 extant species. The phylogenetic position of Zoraptera is poorly understood, but it is generally thought to be closely related to either Paraneoptera (hemipteroid orders: booklice, lice, thrips, and bugs), Dictyoptera (blattoid orders: cockroaches, termites, and mantis), or Embioptera (web spinners). We inferred the phylogenetic position of Zoraptera by analyzing nuclear 18S rDNA sequences, which we aligned according to a secondary structure model. Maximum likelihood and Bayesian analyses both supported a close relationship between Zoraptera and Dictyoptera with relatively high posterior probability. The 18S sequences of Zoraptera exhibited several unusual properties: (1) a dramatically increased substitution rate, which resulted in very long branches; (2) long insertions at helix E23; and (3) modifications of secondary structures at helices 12 and 18.

A tertiary structural element in S box leader RNAs is required for S‐adenosylmethionine‐directed transcription termination

The S box transcription termination control system regulates expression of genes involved in methionine metabolism. Expression of the S box regulon, comprised of 11 transcriptional units in Bacillus subtilis, is induced in response to starvation for methionine. We previously demonstrated that S-adenosylmethionine (SAM) is the molecular effector sensed by the S box leader RNAs during transcription. A secondary structure model for S box leader RNAs was developed based on conservation of primary sequence elements and sequence covariation in helical domains. Covariation of nucleotides in two distantly spaced unpaired regions in the S box leader RNAs suggested that these two domains might interact in the RNA tertiary structure. In this study, site-directed mutagenesis of the covarying residues in two B. subtilis S box leader sequences was employed to test the hypothesis that base-pairing between these regions may be important. The effect of these mutations on in vivo expression, transcription termination in vitro, SAM binding, and leader RNA structure strongly supported the model that interaction between these two regions plays a key role in S box leader function. This provides the first insight into the three-dimensional arrangement of structural elements within the S box RNAs.

Probing the structure of Saccharomyces cerevisiae RNase MRP

In yeast, RNase MRP (mitochondrial RNA processing), a ribonucleoprotein precursor rRNA processing enzyme, possesses one putatively catalytic RNA and ten protein subunits and is highly related to RNase P. Structural analysis of the MRP RNA provides data that closely match a previous secondary-structure model derived from phylogenetic analysis, with the exception of an additional stem. This stem occupies an equivalent position to the P7 stem of RNase P RNA and its inclusion confers on MRP RNA a greater similarity to the core P RNA structure. In vivo studies indicate that the P7-like stem can form, but is not a part of, the active enzyme structure. Stem formation would increase RNA stability in the absence of proteins and our alternative structure may be a valid intermediate species in RNase MRP assembly. Further ongoing studies of this enzyme reveal an extensive network of interactions between subunits and a probable central role for the Pop1, Pop4 and Pop7 subunits.

A Secondary Structural Model of the 28S rRNA Expansion Segments D2 and D3 for Chalcidoid Wasps (Hymenoptera: Chalcidoidea)

We analyze the secondary structure of two expansion segments (D2, D3) of the 28S ribosomal (rRNA)-encoding gene region from 527 chalcidoid wasp taxa (Hymenoptera: Chalcidoidea) representing 18 of the 19 extant families. The sequences are compared in a multiple sequence alignment, with secondary structure inferred primarily from the evidence of compensatory base changes in conserved helices of the rRNA molecules. This covariation analysis yielded 36 helices that are composed of base pairs exhibiting positional covariation. Several additional regions are also involved in hydrogen bonding, and they form highly variable base-pairing patterns across the alignment. These are identified as regions of expansion and contraction or regions of slipped-strand compensation. Additionally, 31 single-stranded locales are characterized as regions of ambiguous alignment based on the difficulty in assigning positional homology in the presence of multiple adjacent indels. Based on comparative analysis of these sequences, the largest genetic study on any hymenopteran group to date, we report an annotated secondary structural model for the D2, D3 expansion segments that will prove useful in assigning positional nucleotide homology for phylogeny reconstruction in these and closely related apocritan taxa.