Primary and secondary structure of the 18 S ribosomal RNA of the insect species Tenebrio molitor

The sequence of the 18 S rRNA of Tenebrio molitor is reported. A detailed secondary structure model for eukaryotic small subunit rRNAs is proposed. The model comprises 48 universal helices that eukaryotic and prokaryotic small subunit rRNAs have in common, plus a number of helices in areas of variable secondary structure. For the central area of the model, an alternative structure is possible, applicable only to eukaryotic small subunit rRNAs. Possibly, small subunit rRNA switched to this alternative conformation after the eukaryotic branch had been established in evolution. Another possibility is that the two conformers represent a dynamic structural switch functioning during the translational activity of the eukaryotic ribosome.     

Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments

Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Δ7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections.     

The nucleotide sequence of the 17S ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin

We have determined the complete sequence of the nuclear gene encoding the small subunit (17 S) rRNA of the ciliated protozoan Tetrahymena thermophila. The gene encodes an RNA molecule which is 1753 nucleotides in length. The sequence of the Tetrahymena small subunit rRNA is homologous to those of other eukaryotes, and the predicted secondary structure for the molecule includes features which are characteristic of eukaryotic small subunit rRNAs. We have also determined the nature of two different mutations in the Tetrahymena 17 S gene which result in resistance to the aminoglycoside antibiotics paromomycin and hygromycin. In each case we have identified a single base change near the 3' end of the rRNA, within a region that is highly evolutionarily conserved in both sequence and secondary structure. Analysis of the effects of these mutations on rRNA structure, and of the impact of these drugs on translation, should help to elucidate the role of the small subunit ribosomal RNA in ribosome function.     

Phenotypic effects of targeted mutations in the small subunit rRNA gene of Tetrahymena thermophila.

Tetrahymena thermophila is an ideal organism with which to study functional aspects of the rRNAs in vivo since the somatic rRNA genes of T. thermophila can be totally replaced by cloned copies introduced via microinjection. In this study, we made small insertions into seven sites within the small subunit rRNA gene and observed their phenotypic effects on transformed cells. Two mutated genes coding for rRNA (rDNAs), both of which bear insertions in highly conserved sequences, failed to transform and are therefore believed to produce nonfunctional rRNAs. Three other altered rDNAs produce functional rRNAs that can substitute for most or all of the cellular rRNA. Two of these bear insertions in highly variable regions, and, surprisingly, the other has an insertion in a region that is well conserved for both sequence and secondary structure among eucaryotes. In addition, two other insertions appear to destabilize rRNAs that contain them. Our findings make predictions concerning the positions of some of these sites within the tertiary structure of the small ribosomal subunit and thus serve as an in vivo test of the existing tertiary structure models for the small subunit rRNA. Our results are in good agreement with expectations based on sequence comparison and in vitro work.     

Repeated parallel evolution of minimal rRNAs revealed from detailed comparative analysis

The concept of a minimal ribosomal RNA-containing ribosome, a structure with a minimal set of elements capable of providing protein biosynthesis, is essential for understanding this fundamental cellular process. Nematodes and trypanosomes have minimal mitochondrial rRNAs and detailed reconstructions of their secondary structures indicate that certain conserved helices have been lost in these taxa. In contrast, several recent studies on acariform mites have argued that minimal rRNAs may evolve via shortening of secondary structure elements but not the loss of these elements as shown for trypanosomes and nematodes. Based on extensive structural analysis of chelicerate arthropods, we demonstrate that extremely short rRNAs of acariform mites share certain structural modifications with nematodes and trypanosomes: loss of helices of the GTPase region and divergence in the evolutionarily conserved connecting loop between helices H1648 and H1764 of the large subunit rRNA. These highly concerted parallel modifications indicate that minimal rRNAs were generated under the strong selection that favored or tolerated reductions of helices in particular locations while maintaining the functionality of the rRNA molecules throughout evolution. We also discuss potential evolution of minimal rRNAs and atypical transfer RNAs.     

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.     

Estimating Substitution Rates in Ribosomal RNA Genes

A model is introduced describing nucleotide substitution in ribosomal RNA (rRNA) genes. In this model, substitution in the stem and loop regions of rRNA is modeled with 16- and four-state continuous time Markov chains, respectively. The mean substitution rates at nucleotide sites are assumed to follow gamma distributions that are different for the two types of regions. The simplest formulation of the model allows for explicit expressions for transition probabilities of the Markov processes to be found. These expressions were used to analyze several 16S-like rRNA genes from higher eukaryotes with the maximum likelihood method. Although the observed proportion of invariable sites was only slightly higher in the stem regions, the estimated average substitution rates in the stem regions were almost two times as high as in the loop regions. Therefore, the degree of site heterogeneity of substitution rates in the stem regions seems to be higher than in the loop regions of animal 16S-like rRNAs due to presence of a few rapidly evolving sites. The model appears to be helpful in understanding the regularities of nucleotide substitution in rRNAs and probably minimizing errors in recovering phylogeny for distantly related taxa from these genes.     

A comparison of the yeast and rabbit 80 S ribosome reveals the topology of the nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments

Protein synthesis in eukaryotes is mediated by both cytoplasmic and membrane-bound ribosomes. During the co-translational translocation of secretory and membrane proteins, eukaryotic ribosomes dock with the protein conducting channel of the endoplasmic reticulum. An understanding of these processes will require the detailed structure of a eukaryotic ribosome. To this end, we have compared the three-dimensional structures of yeast and rabbit ribosomes at 24 A resolution. In general, we find that the active sites for protein synthesis and translocation have been highly conserved. It is interesting that a channel was visualized in the neck of the small subunit whose entrance is formed by a deep groove. By analogy with the prokaryotic small subunit, this channel may provide a conserved portal through which mRNA is threaded into the decoding center. In addition, both the small and large subunits are built around a dense tubular network. Our analysis further suggests that the nascent chain exit tunnel and the docking surface for the endoplasmic reticulum channel are formed by this network. We surmise that many of these features correspond to rRNA, based on biochemical and structural data.Ribosomal function is critically dependent on the specific association of small and large subunits. Our analysis of eukaryotic ribosomes reveals four conserved inter-subunit bridges with a geometry similar to that found in prokaryotes. In particular, a double-bridge connects the small subunit platform with the interface canyon on the large subunit. Moreover, a novel bridge is formed between the platform and the base of the L1 domain. Finally, size differences between mammalian and yeast large subunit rRNAs have been correlated with five expansion segments that form two large spines and three extended fingers. Overall, we find that expansion segments within the large subunit rRNA have been incorporated at positions distinct from the active sites for protein synthesis and translocation.     

The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions

Polymerase chain reaction conditions were established for the in vitro amplification of eukaryotic small subunit ribosomal (16S-like) rRNA genes. Coding regions from algae, fungi, and protozoa were amplified from nanogram quantities of genomic DNA or recombinant plasmids containing rDNA genes. Oligodeoxynucleotides that are complementary to conserved regions at the 5' and 3' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis. The fidelity of synthesis for the amplification products was evaluated by comparisons with sequences of previously reported rRNA genes or with primer extension analyses of rRNAs. Fewer than one error per 2000 positions were observed in the amplified rRNA coding region sequences. The primary structure of the 16S-like rRNA from the marine diatom, Skeletonema costatum, was inferred from the sequence of its in vitro amplified coding region.     

An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs.     

Role of the 5.8S rRNA in ribosome translocation.

Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that this RNA plays an important role in eukaryotic ribosome function. Mutations in the 5. 8S rRNA can inhibit cell growth and compromise protein synthesis in vitro . Polyribosomes from cells expressing these mutant 5.8S rRNAs are elevated in size and ribosome-associated tRNA. Cell free extracts from these cells also are more sensitive to antibiotics which act on the 60S ribosomal subunit by inhibiting elongation. The extracts are especially sensitive to cycloheximide and diphtheria toxin which act specifically to inhibit translocation. Studies of ribosomal proteins show no reproducible changes in the core proteins, but reveal reduced levels of elongation factors 1 and 2 only in ribosomes which contain large amounts of mutant 5.8S rRNA. Polyribosomes from cells which are severely inhibited, but contain little mutant 5.8S rRNA, do not show the same reductions in the elongation factors, an observation which underlines the specific nature of the change. Taken together the results demonstrate a defined and critical function for the 5.8S rRNA, suggesting that this RNA plays a role in ribosome translocation.     

Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.     

Saturation Mutagenesis of 5S rRNA in Saccharomyces cerevisiae

rRNAs are the central players in the reactions catalyzed by ribosomes, and the individual rRNAs are actively involved in different ribosome functions. Our previous demonstration that yeast 5S rRNA mutants (called mof9) can impact translational reading frame maintenance showed an unexpected function for this ubiquitous biomolecule. At the time, however, the highly repetitive nature of the genes encoding rRNAs precluded more detailed genetic and molecular analyses. A new genetic system allows all 5S rRNAs in the cell to be transcribed from a small, easily manipulated plasmid. The system is also amenable for the study of the other rRNAs, and provides an ideal genetic platform for detailed structural and functional studies. Saturation mutagenesis reveals regions of 5S rRNA that are required for cell viability, translational accuracy, and virus propagation. Unexpectedly, very few lethal alleles were identified, demonstrating the resilience of this molecule. Superimposition of genetic phenotypes on a physical map of 5S rRNA reveals the existence of phenotypic clusters of mutants, suggesting that specific regions of 5S rRNA are important for specific functions. Mapping these mutants onto the Haloarcula marismortui large subunit reveals that these clusters occur at important points of physical interaction between 5S rRNA and the different functional centers of the ribosome. Our analyses lead us to propose that one of the major functions of 5S rRNA may be to enhance translational fidelity by acting as a physical transducer of information between all of the different functional centers of the ribosome.     

Structure of the Large Subunit rDNA from a Diatom, and Comparison Between Small and Large Subunit Ribosomal RNA for Studying Stramenopile Evolution

The aim of this study was to compare the usefulness of complete small and large subunit rRNA, and a combination of both molecules, for reconstructing stramenopile evolution. To this end, phylogenies from species of which both sequences are known Acre constructed with the neighbor-joining, maximum parsimony, and maximum likelihood methods. Also the use of structural features of the rRNAs was evaluated. The large subunit rRNA from the diatom Skeletonema pseudocostatum was sequenced in order to have a more complete taxon sampling, and a group I intron was identified. Our results indicated that heterokont algae are monophyletic, with diatoms diverging first. However, as the analysis was restricted to a particular data set containing merely six taxa, the outcome has limited value for elucidating stramenopile relationships. On the other hand, this approach permits comparison of the performance of both rRNA molecules without interference from other factors, such as a different species selection for each molecule. For the taxa used, the large subunit rRNA clearly contained more phylogenetic information than the small subunit rRNA. Although this result can definitely not be generalized and depends on the phvlogeny to be studied, in some cases determining complete large subunit rRNA sequences certainly seems worthwhile.     

Putative secondary structures of unusually long strepsipteran SSU rRNAs and its phylogenetic implications.

We constructed the putative secondary structures of the small subunit rRNAs (SSU rRNA) from three strepsipteran insects. The primary sequences of the strepsipteran SSU rRNAs are unusually long due to unique and long insertions. In spite of these insertions, the basic shapes of their secondary structures are well maintained as shown in those of other eukaryotes, because these insertions appear mainly in the variable regions. The secondary structures for the V1, V3, V5, V8, and V9 regions are well conserved, even though the primary structures of V1, V5, and V8 regions are quite variable. However, the predicted secondary structures for the V2, V4, and V7 regions are quite different from those of other insects. In the V4 and V7 regions, helices specific to the Strepsiptera exist. These helices have not been reported in other organisms so far. Similarly, four eukaryotic specific helices (E8-1, E10-2, E23-4 and E45-1) not reported in insects exist in the V2, V4, and V8 regions. These helices are formed by the inserted sequences. The secondary structures of the expanded segments of the strepsipteran SSU rRNA were applied to infer the phylogenetic position of Strepsiptera, one of the most enigmatic problems in insect phylogeny. Only the secondary structure of the V7 region showed the weak Strepsiptera/Diptera sister-group relationship.     

Nucleotide sequence of 5S ribosomal RNA from rainbow trout (Salmo gairdnerii) liver.

Staring from low molecular weight RNA obtained from rainbow trout (Salmo gairdnerii) liver, 5S ribosomal RNA (rRNA) was highly purified by successive chromatography on columns of DEAE-Sephadex A50 and Sephadex G100. Products of complete and partial digestions on this RNA with pancreatic ribonuclease (RNase A) [EC 3.1.4.22] and RNase T [EC 3.1.4.8] were isolated and sequenced by conventional and high-performance liquid chromatography (HPLC) procedures. The nucleotide sequence of this RNA thus established was compared with those of five other vertebrae 5S rRNAs, and the rates of base substitution per site per year were found to be nearly constant in these RNAs. The analyses of the partial digests of the trout 5S rRNA revealed several sites susceptible to RNase attack, which could be accounted for by the secondary structure model for eukaryotic 5S rRNAs proposed by Nishikawa and Takemura (1).     

Secondary structure of the Dictyostelium discoideum small subunit ribosomal RNA.

We have used comparative analyses of prokaryotic and eukaryotic small subunit ribosomal RNAs to deduce a secondary structure for the Dictyostelium discoideum 18S rRNA. Most of the duplex regions are evolutionarily conserved in all organisms. We have taken advantage of the variation to the D. discoideum sequence (relative to the yeast and frog 19S rRNAs) to identify additional helical regions which are common to the eukaryotic 18S rRNAs.