Structural analysis of prokaryotic and eukaryotic 5S rRNAs by RNase H

The availabilities of single-stranded 5S rRNA regions c, d and d' for base pairing interactions were analyzed by using synthetic DNA oligomers. Hybrid formation was detected by the endonucleolytical mode of the RNA-DNA specific action of RNase H. Provided that the hybrid interaction involved 6 successive base pairs, 5S rRNA loop c nucleotides 42-47 displayed accessibility in Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus 5S rRNAs as well as in eukaryotic 5S rRNAs from Saccharomyces carlsbergensis, Rattus rattus and Equisetum arvense. Investigating eubacterial 5S rRNA regions d and d' (nucleotides 71-76 and 99-105, respectively), susceptibility was observed in E. coli 5S rRNA which, however, decreases in B. stearothermophilus and even more so in T. thermophilus 5S rRNA. For additional evaluation of the data obtained by RNase H cleavage, association constants of the hexanucleotides were determined by equilibrium dialysis at 4 degrees C for B. stearothermophilus 5S rRNA. The results obtained reveal that nucleotides 36-41 of B. stearothermophilus 5S rRNA are inaccessible for Watson-Crick interaction, which suggests that this part of loop c is in a structurally constrained configuration, or buried in the tertiary structure or involved in tertiary interactions.     

Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules

5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator.     

Molecular coevolution among cryptically simple expansion segments of eukaryotic 26S/28S rRNAs

The set of "expansion segments" of any eukaryotic 26S/28S ribosomal RNA (rRNA) gene is responsible for the bulk of the difference in length between the prokaryotic 23S rRNA gene and the eukaryotic 26S/28S rRNA gene. The expansion segments are also responsible for interspecific fluctuations in length during eukaryotic evolution. They show a consistent bias in base composition in any species; for example, they are AT rich in Drosophila melanogaster and GC rich in vertebrate species. Dot-matrix comparisons of sets of expansion segments reveal high similarities between members of a set within any 28S rRNA gene of a species, in contrast to the little or spurious similarity that exists between sets of expansion segments from distantly related species. Similarities among members of a set of expansion segments within any 28S rRNA gene cannot be accounted for by their base-compositional bias alone. In contrast, no significant similarity exists within a set of "core" segments (regions between expansion segments) of any 28S rRNA gene, although core segments are conserved between species. The set of expansion segments of a 26S/28S gene is coevolving as a unit in each species, at the same time as the family of 28S rRNA genes, as a whole, is undergoing continual homogenization, making all sets of expansion segments from all ribosomal DNA (rDNA) arrays in a species similar in sequence. Analysis of DNA simplicity of 26S/28S rRNA genes shows a direct correlation between significantly high relative simplicity factors (RSFs) and sequence similarity among a set of expansion segments. A similar correlation exists between RSF values, overall rDNA lengths, and the lengths of individual expansion segments. Such correlations suggest that most length fluctuations reflect the gain and loss of simple sequence motifs by slippage-like mechanisms. We discuss the molecular coevolution of expansion segments, which takes place against a background of slippage-like and unequal crossing-over mechanisms of turnover that are responsible for the accumulation of interspecific differences in rDNA sequences.      

500-MHz proton homonuclear Overhauser evidence for additional base pair in the common arm of eukaryotic ribosomal 5S RNA: wheat germ

A "common-arm" fragment from wheat germ (Triticum aestivum) 5S RNA has been produced by enzymatic cleavage with RNase T1 and sequenced via autoradiography of electrophoresis gels for the end-labeled fragments obtained by further RNase T1 partial digestion. The existence, base pair composition, and base pair sequence of the common arm are demonstrated for the first time by means of proton 500-MHz nuclear magnetic resonance. From Mg2+ titration, temperature variation, ring current calculations, sequence comparisons, and proton homonuclear Overhauser enhancement experiments, additional base pairs in the common arm of the eukaryotic 5S RNA secondary structure are detected. Two base pairs, G41 X C34 and A42 X U33 in the hairpin loop, could account for the lack of binding between the conserved GAAC segment of 5S RNA and the conserved Watson-Crick-complementary GT psi C segment of tRNAs.     

Differentiation of oocyte- and somatic-type 5S rRNAs in animals

In some amphibians and bony fishes, oocyte- and somatic-type 5S rRNA genes are expressed differently in oocytes and somatic cells. In order to determine at what stage of animal evolution this differential expression system appeared and how it is regulated, the sequences of oocyte and somatic 5S rRNAs from three invertebrates (sea urchin, sea hare, and silkworm) and two vertebrates (lamprey and chick) were analyzed. It was found that the oocyte 5S rRNA from lamprey consists of two components, while its somatic 5S rRNA consists of only one. In other animals, such differential expression of 5S rRNA in oocytes and somatic cells was not seen. A phylogenetic tree of 63 animal 5S rRNAs was constructed by means of the parsimony method, and the evolution of oocyte and somatic-type 5S rRNAs was discussed.     

The calculation of plant 5S rRNAs secondary structure

Using commercially available computer software package for ribonucleic acid (RNA) secondary structure analysis we calculated the free energy (delta G) of all higher plant 5S rRNA species. To gain insight into the relation between structure (nucleotide sequence) and free energy we generated point mutants of plant 5S rRNA and calculated their secondary structure. This analysis permitted to identify single sites which affect the stability and conformation of RNA molecule. Furthermore, the calculated data were compared with the electrophoretic mobility of 5S rRNA on polyacrylamide gels.     

Different mechanisms for pseudouridine formation in yeast 5S and 5.8S rRNAs

The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Psis) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Psi formation in 5S and 5.8S rRNA in which the unassigned Psis occur. We show that while the formation of the Psi in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Psi in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Psi in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.     

Nucleotide sequences of 5S rRNAs from four jellyfishes.

The nucleotide sequences of 5S rRNAs from four jellyfishes, Spirocodon saltatrix, Nemopsis dofleini, Aurelia aurita and Chrysaora quinquecirrha have been determined. The sequences are highly similar to each other. A fairly high similarity was also found between these jellyfishes and a sea anemone, Anthopleura japonica.     

Secondary structure and domain architecture of the 23S and 5S rRNAs

We present a de novo re-determination of the secondary (2°) structure and domain architecture of the 23S and 5S rRNAs, using 3D structures, determined by X-ray diffraction, as input. In the traditional 2° structure, the center of the 23S rRNA is an extended single strand, which in 3D is seen to be compact and double helical. Accurately assigning nucleotides to helices compels a revision of the 23S rRNA 2° structure. Unlike the traditional 2° structure, the revised 2° structure of the 23S rRNA shows architectural similarity with the 16S rRNA. The revised 2° structure also reveals a clear relationship with the 3D structure and is generalizable to rRNAs of other species from all three domains of life. The 2° structure revision required us to reconsider the domain architecture. We partitioned the 23S rRNA into domains through analysis of molecular interactions, calculations of 2D folding propensities and compactness. The best domain model for the 23S rRNA contains seven domains, not six as previously ascribed. Domain 0 forms the core of the 23S rRNA, to which the other six domains are rooted. Editable 2° structures mapped with various data are provided (http://apollo.chemistry.gatech.edu/RibosomeGallery).     

In vitro incorporation of eubacterial, archaebacterial and eukaryotic 5S rRNAs into large ribosomal subunits of Bacillus stearothermophilus.

Bacillus stearothermophilus large ribosomal subunits were reconstituted in the presence of 5S rRNAs from different origins and tested for their biological activities. The results obtained have shown that eubacterial and archaebacterial 5S rRNAs can easily substitute for B. stearothermophilus 5S rRNA in the reconstitution, while eukaryotic 5S rRNAs yield ribosomal subunits with reduced biological activities. From our results we propose an interaction between nucleotides 42-47 of 5S rRNA and nucleotides 2603-2608 of 23S rRNA during the assembly of the 50S ribosomal subunit. Other experiments with eukaryotic 5.8S rRNAs reveal, if at all, a very low incorporation of these RNA species into the reconstituted ribosomes.     

Secondary structure and phylogeny of Staphylococcus and Micrococcus 5S rRNAs

Nucleotide sequences of 5S rRNAs from four bacteria, Staphylococcus aureus Smith (diffuse), Staphylococcus epidermidis ATCC 14990, Micrococcus luteus ATCC 9341 and Micrococcus luteus ATCC 4698, were determined. The secondary structural models of S. aureus and S. epidermidis sequences showed characteristics of the gram-positive bacterial 5S rRNA (116-N type [H. Hori and S. Osawa, Proc. Natl. Acad. Sci. U.S.A. 76:381-385, 1979]). Those of M. luteus ATCC 9341 and M. luteus ATCC 4698 together with that of Streptomyces griseus (A. Simoncsits, Nucleic Acids Res. 8:4111-4124, 1980) showed intermediary characteristics between the gram-positive and gram-negative (120-N type [H. Hori and S. Osawa, 1979]) 5S rRNAs. This and previous studies revealed that there exist at least three major groups of eubacteria having distinct 5S rRNA and belonging to different stems in the 5S rRNA phylogenic tree.     

Unbalanced ribosome assembly in Saccharomyces cerevisiae expressing mutant 5 S rRNAs.

Mutant 5 S rRNA genes were expressed in Saccharomyces cerevisiae to further define the function of the ribosomal 5 S RNA. RNA synthesis and utilization were assayed using previously constructed markers which have been shown to be functionally neutral and easily detected by gel electrophoresis. Most mutations were found not to affect the growth rate because they were poorly expressed or could be accommodated effectively in the ribosomal structure. Two of the mutants, Y5A99U56U57 and Y5U90i5 adversely affected cell growth as well as protein synthesis in vitro. Polyribosome profiles in both of these mutants were substantially shorter, and an analysis of the ribosomal subunit composition revealed a significant imbalance with a 25-35% excess in 40 S subunits. Kinetic analyses of RNA labeling indicated very low cellular levels of mutant RNA either because it was poorly expressed (Y5U90i5) or rapidly degraded before being incorporated into mature 60 subunits (Y5A99U56U57). The results suggest that the 5 S RNA is required for the assembly of stable ribosomal 60 S subunits and raise the possibility that this RNA or, more likely, its corresponding ribonucleoprotein complex is critical for subunit assembly or even RNA processing.     

MIAH: automatic alignment of eukaryotic SSU rRNAs.

SUMMARY: MIAH is a WWW server for the automatic alignment of new eukaryotic SSU rRNA sequences to an existing alignment of 1500 sequences. AVAILABILITY: http://chah.ucc.ie/MIAH Contact :     

Unbalanced regulation of the ribosomal 5 S RNA-binding protein in Saccharomyces cerevisiae expressing mutant 5 S rRNAs.

A gene encoding the 5 S rRNA-binding protein (YL3) in yeast (Saccharomyces cerevisiae) was further characterized with respect to its chromosomal localization, the controlling sequence regions, and the influence of 5 S rRNA gene expression. Sequence and chromosome blot analyses localized the gene on chromosome XVI immediately downstream of a cytochrome oxidase assembly gene, COXII. S1 nuclease protection studies identified two major initiation sites, 20 and 65 nucleotides upstream of the coding sequence, and a single polyadenylation site, 98 nucleotides downstream of the stop codon. Northern blot analyses and S1 nuclease protection indicated a normal pattern of gene regulation in media supporting alternate rates of growth, but significantly unbalanced regulation was observed in the presence of mutant 5 S rRNA genes which under-produce RNA and result in reduced growth rates. The results suggest a co-ordinating regulatory mechanism which maintains appropriate levels of 5 S rRNA-protein complex; an internal control region-like sequence in the upstream region of the YL3 gene is consistent with this feedback mechanism.     

Dictyostelium 17S, 25S, and 5S rDNAs lie within a 38,000 base pair repeated unit.

Mapping with the restriction enzymes Sal 1 and R1 has generated a picture of the organization of Dictyostelium ribosomal DNA. The DNA which codes for 17S and 25S ribosomal RNAs is located within a stretch of repetitive DNA at least 38,000 base pairs long. This repeated unit includes 5S DNA, linked to 25S DNA. Two techniques were especially useful in the mapping: "cloning" 14S + 25S DNA on the plasmid pMB9 to amplify individual R1 fragments, and digesting DNA with R1 in the presence of the antibiotic distamycin A to produce specific partial digestion products.     

The nucleotide sequences of 5S rRNAs from three ciliated protozoa.

The nucleotide sequences of 5S rRNAs from three ciliated protozoa, Paramecium tetraurelia, Tetrahymena thermophila and Blepharisma japonicum have been determined. All of them are 120 nucleotides long and the sequence of probable tRNA binding site of position 41-44 is GAAC which is characteristic of the plant 5S rRNAs. The sequence similarity percents are 87% (Paramecium/Tetrahymena), 86% (Paramecium/Blepharisma) and 79% (Tetrahymena/Blepharisma), suggesting a close relationship of these three ciliates.     

Pea aphid symbiont relationships established by analysis of 16S rRNAs

The pea aphid (Acyrthosiphon pisum Harris) harbors two morphologically distinct procaryotic intracellular symbionts. The genes for the 16S rRNA from these symbionts have been cloned and sequenced. Comparisons with sequences of 16S rRNAs from selected procaryotes indicate that the two symbionts are evolutionarily distinct from each other and are members of the gamma-3 subdivision of the class Proteobacteria. One of the symbionts is a member of the family Enterobacteriaceae, while the other constitutes a lineage distinct from these organisms. Both symbionts appear to have only one copy of their rRNA operon.     

Euglena gracilis chloroplast ribosomal RNA transcription units. Nucleotide sequence polymorphism in 5 S rRNA genes and 5 S rRNAs

The three tandemly repeated ribosomal RNA operons from the chloroplast genome of Euglena gracilis Klebs, Pringsheim Strain Z each contain a 5 S rRNA gene distal to the 23 S rRNA gene (Gray, P.W., and Hallick, R.B. (1979) Biochemistry 18, 1820-1825). We have cloned two distinct 5 S rRNA genes, and determined the DNA sequence of the genes, their 5'- and 3'-flanking sequences, and the 3'-end of the adjacent 23 S rRNA genes. The two genes exhibit sequence polymorphism at five bases within the "procaryotic loop" coding region, as well as internal restriction endonuclease site heterogeneity. These restriction endonuclease site polymorphisms are evident in chloroplast DNA, and not just the cloned examples of 5 S genes. Chloroplast 5 S rRNA was isolated, end labeled, and sequenced by partial enzymatic degradation. The same polymorphisms found in 5 S rDNA are present in 5 S rRNA. Therefore, both types of 5 S rRNA genes are transcribed and are present in chloroplast ribosomes.