Robin R. Gutell

The Comparative RNA Web (CRW) Site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs

BackgroundComparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields.ResultsWe have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems.ConclusionsThis online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site.

Comparative Anatomy of 16-S-like Ribosomal RNA

Publisher Summary This chapter examines the range of the variation of secondary structure among the 16-S-like rRNAs. This brings into a larger structural context a recent detailed analysis of the individual helical elements and provides a basis for an accurate alignment of the corresponding regions of different primary structures. Computer-assisted comparative is used in the analysis of aligned sequences to describe the pattern of phylogenetic conservation for each nucleotide position in 16-S rRNA. A search for matching patterns among unpaired positions in the RNA chain then produces a list of candidates for potential base–base tertiary interactions. The completion of nucleotide sequences for 34 16-S-like rRNAs includes 4 eubacteria, 4 chloroplasts, 12 mitochondria, 4 archaebacteria, and 10 eukaryotes. Secondary structure models for these molecules have been developed in the course of refinement of the E. coli model, and have been used to arrive at improved sequence alignments for the 16-S-like rRNAs. Schematic drawings of (1) eubacterial, (2) archaebacterial, (3) eukaryotic cytoplasmic, (4) plant mitochondrial, (5) fungal mitochondrial, and (6) mammalian mitochondrial structures are shown in the chapter.

Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli.

Abstract We have constructed recombinant plasmids containing the entire Escherichia coli rrnB ribosomal RNA operon and segments thereof. Cloning of the 7.5-kb BamHI fragment, from λrifd18 which contains this operon, in plasmid vectors pBR 313 or pBR 322 is described. The 3.2-kb Eco RI Bam HI fragment containing the 3′ two-thirds of the 23 S rRNA gene, the 5S rRNA gene, and the terminator region has been cloned separately in pBR 313. As the nucleotide sequences of pBR 322 and the 7.5-kb fragment carrying the rrnB operon have been established, the entire 11.9-kb sequence of pKK 3535 is now known. This makes possible precise rearrangements and site-specific alterations of the ribosomal RNA operon; thus, pKK 3535 becomes a powerful tool for studies such as initiation and termination of transcription, processing of rRNA precursors, and investigations of the structure, function, and assembly of the ribosome itself. A detailed physical map of pKK 3535 is presented.

The accuracy of ribosomal RNA comparative structure models.

The determination of the 16S and 23S rRNA secondary structure models was initiated shortly after the first complete 16S and 23S rRNA sequences were determined in the late 1970s. The structures that are common to all 16S rRNAs and all 23S rRNAs were determined using comparative methods from the analysis of thousands of rRNA sequences. Twenty-plus years later, the 16S and 23S rRNA comparative structure models have been evaluated against the recently determined high-resolution crystal structures of the 30S and 50S ribosomal subunits. Nearly all of the predicted covariation-based base pairs, including the regular base pairs and helices, and the irregular base pairs and tertiary interactions, were present in the 30S and 50S crystal structures.

Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements

As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)‐encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non‐conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome, we expect our report eventually to shed light on the evolution of the hymenopteran genome within higher insects, particularly regarding the relative maintenance of conserved rDNA genes, related variable spacer regions and retrotransposable elements.

Phylogenetic Analyses of Basal Angiosperms Based on Nine Plastid, Mitochondrial, and Nuclear Genes

DNA sequences of nine genes (plastid: atpB, matK, and rbcL; mitochondrial: atp1, matR, mtSSU, and mtLSU; nuclear: 18S and 26S rDNAs) from 100 species of basal angiosperms and gymnosperms were analyzed using parsimony, Bayesian, and maximum likelihood methods. All of these analyses support the following consensus of relationships among basal angiosperms. First, Amborella, Nymphaeaceae, and Austrobaileyales are strongly supported as a basal grade in the angiosperm phylogeny, with either Amborella or Amborella and Nymphaeales as sister to all other angiosperms. An examination of nucleotide substitution patterns of all nine genes ruled out any possibility of analytical artifacts because of RNA editing and GC‐content bias in placing these taxa at the base of the angiosperm phylogeny. Second, Magnoliales are sister to Laurales and Piperales are sister to Canellales. These four orders together constitute the magnoliid clade. Finally, the relationships among Ceratophyllum, Chloranthaceae, monocots, magnoliids, and eudicots are resolved in different ways in various analyses, mostly with low support. Our study indicates caution in total evidence approaches in that some of the genes employed (e.g., mtSSU, mtLSU, and nuclear 26S rDNA) added signal that conflicted with the other genes in resolving certain parts of the phylogenetic tree.

Modeling a Minimal Ribosome Based on Comparative Sequence Analysis

We have determined the three-dimensional organization of ribosomal RNAs and proteins essential for minimal ribosome function. Comparative sequence analysis identifies regions of the ribosome that have been evolutionarily conserved, and the spatial organization of conserved domains is determined by mapping these onto structures of the 30S and 50S subunits determined by X-ray crystallography. Several functional domains of the ribosome are conserved in their three-dimensional organization in the Archaea, Bacteria, Eucaryotic nuclear, mitochondria and chloroplast ribosomes. In contrast, other regions from both subunits have shifted their position in three-dimensional space during evolution, including the L11 binding domain and the alpha-sarcin-ricin loop (SRL). We examined conserved bridge interactions between the two ribosomal subunits, giving an indication of which contacts are more significant. The tRNA contacts that are conserved were also determined, highlighting functional interactions as the tRNA moves through the ribosome during protein synthesis. To augment these studies of a large collection of comparative structural models sampled from all major branches on the phylogenetic tree, Caenorhabditis elegans mitochondrial rRNA is considered individually because it is among the smallest rRNA sequences known. The C.elegans model supports the large collection of comparative structure models while providing insight into the evolution of mitochondrial ribosomes.

ITS secondary structure derived from comparative analysis: implications for sequence alignment and phylogeny of the Asteraceae

An RNA secondary structure model is presented for the nuclear ribosomal internal transcribed spacers (ITS) based on comparative analysis of 340 sequences from the angiosperm family Asteraceae. The model based on covariation analysis agrees with structural features proposed in previous studies using mainly thermodynamic criteria and provides evidence for additional structural motifs within ITS1 and ITS2. The minimum structure model suggests that at least 20% of ITS1 and 38% of ITS2 nucleotide positions are involved in base pairing to form helices. The sequence alignment enabled by conserved structural features provides a framework for broadscale molecular evolutionary studies and the first family-level phylogeny of the Asteraceae based on nuclear DNA data. The phylogeny based on ITS sequence data is very well resolved and shows considerable congruence with relationships among major lineages of the family suggested by chloroplast DNA studies, including a monophyletic subfamily Asteroideae and a paraphyletic subfamily Cichorioideae. Combined analyses of ndhF and ITS sequences provide additional resolution and support for relationships in the family.

Structure of the mammalian 80S ribosome at 8.7 A resolution.

In this paper, we present a structure of the mammalian ribosome determined at approximately 8.7 A resolution by electron cryomicroscopy and single-particle methods. A model of the ribosome was created by docking homology models of subunit rRNAs and conserved proteins into the density map. We then modeled expansion segments in the subunit rRNAs and found unclaimed density for approximately 20 proteins. In general, many conserved proteins and novel proteins interact with expansion segments to form an integrated framework that may stabilize the mature ribosome. Our structure provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation. Finally, details are presented for intersubunit bridges that are specific to the eukaryotic ribosome. We suggest that these bridges may help reset the conformation of the ribosome to prepare for the next cycle of chain elongation.

Morphological, molecular, and phylogenetic characterization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera.

ABSTRACT. Nosema ceranae, a microsporidian parasite originally described from Apis cerana, has been found to infect Apis melllifera and is highly pathogenic to its new host. In the present study, data on the ultrastructure of N. ceranae, presence of N. ceranae‐specific nucleic acid in host tissues, and phylogenetic relationships with other microsporidia species are described. The ultrastructural features indicate that N. ceranae possesses all of the characteristics of the genus Nosema. Spores of N. ceranae measured approximately 4.4 × 2.2 μm on fresh smears. The number of coils of the polar filament inside spores was 18–21. Polymerase chain reaction (PCR) signals specific for N. ceranae were detected not only in the primary infection site, the midgut, but also in the tissues of hypopharyngeal glands, salivary glands, Malpighian tubules, and fat body. The detection rate and intensity of PCR signals in the fat body were relatively low compared with other examined tissues. Maximum parsimony analysis of the small subunit rRNA gene sequences showed that N. ceranae appeared to be more closely related to the wasp parasite, Nosema vespula, than to N. apis, a parasite infecting the same host.