The nucleotide sequence of the cytoplasmic 5S rRNA from the horsetail, Equisetum arvense

Using 3'- and 5'-end labelling sequencing techniques, the following sequence for the cytoplasmic 5S rRNA of the horsetail Equisetum arvense could be determined: (sequence in text). This sequence exhibits all features expected for higher plant cytoplasmic 5S rRNAs, and can be fitted to the secondary structure model for 5S rRNA proposed by De Wachter et al. (15).     

Nucleotide sequence and thermal property of 5S rRNA from the elder aphid. Acyrthosiphon magnoliae.

The nucleotide sequence of 5S rRNA from the elder aphid. Acyrthosiphon magnoliae was determined by using postlabeling sequencing techniques. The aphid 5S rRNA consists of 120 nucleotides and the sequence differs from those of Bombyx and Drosophila 5S rRNAs in 14 and 16 positions, respectively. A secondary structure model based on the sequence has two distinctive features : the helix I is shorter and the total free energy lower. Judging from the thermal profile, the aphid 5S rRNA likely assumes a conformation somewhat different from those of the other two insects.     

The Secondary Structure of Mammalian Mitochondrial 16S rRNA Molecules: Refinements Based on a Comparative Phylogenetic Approach

Eighty six complete 16S ribosomal RNA (rRNA) gene sequences representing every mammalian order (one monotreme, 33 marsupials, 52 placentals) were employed to establish a core secondary structure model for mammalian 16S rRNA. Starting with the Gutell et al. (1993) and De Rijk et al. (1999) models, we used the criteria of potential base-pairing and positional covariance to make refinements in these models for mammalian 16S rRNA molecules. Our results suggest a mammalian secondary structure model with deletions as well as additions to the Gutell et al. (1993) and De Rijk et al. (1999) models for cow. We recognize 53 stems, 41 of which show at least some positional covariance within Mammalia. In addition, we recognize four tertiary interactions. Stems and loops have distinctly different properties, including base composition and relative substitution rates. Accounting for these differences results in improved models of sequence evolution.     

Probing the conformation of 18S rRNA in yeast 40S ribosomal subunits with kethoxal

Yeast 40S ribosomal subunits have been reacted with kethoxal to probe the conformation of 18S rRNA. Over 130 oligonucleotides were isolated by diagonal electrophoresis and sequenced, allowing identification of 48 kethoxal-reactive sites in the 18S rRNA chain. These results generally support a secondary structure model for 18S rRNA derived from comparative sequence analysis. Significant reactivity at positions 1436 and 1439, in a region shown to be base paired by comparative analysis, lends support to the earlier suggestion [Chapman, N.M., & Noller, H.F. (1977) J. Mol. Biol 109, 131-149] that part of the 3'-major domain of 16S-like rRNAs may undergo a biologically significant conformational rearrangement. Modification of positions in 18S rRNA analogous to those previously found for Escherichia coli 16S rRNA argues for extensive structural homology between 30S and 40S ribosomal subunits, particularly in regions thought to be directly involved in translation.     

The structure of the 5 S ribosomal RNA of a member of the phylum of green non-sulfur bacteria and relatives

The 5 S rRNA sequence was determined for the bacterium Herpetosiphon strain Senghas Wie 2. It is the first 5 S RNA sequence reported for a member of the eubacterial phylum defined by green non-sulfur bacteria. The sequence fits into a consensus secondary structure model for eubacterial 5 S RNA. At four positions, the sequence shows substitutions with respect to strongly conserved nucleotides found in other hitherto examined eubacterial 5 S RNAs.     

Structural studies on site-directed mutants of domain 3 of Xenopus laevis oocyte 5 S ribosomal RNA

Base substitutions have been introduced into the highly conserved sequences of loops D and E within domain 3 of Xenopus laevis oocyte 5 S rRNA. The effects of these mutations on the solution structure of this 5 S rRNA have been studied by means of probing with nucleases, and with chemical reagents under native and semi-denaturing conditions. The data obtained with these mutants support the graphic model of Xenopus oocyte 5 S rRNA proposed by Westhof et al. In particular, our results rule out the existence of long-range base-pairing interactions between loop C and either loop D or loop E. The data also confirm that loops D and E in the wild-type 5 S RNA adopt unusual secondary structures and illustrate the importance of nucleotide sequence in the formation of intrinsic local loop conformations via non-canonical base-pairs and specific base-phosphate contacts. Consistent with this conclusion is our observation that the domain 3 fragment of Xenopus oocyte 5 S rRNA adopts the same conformation as the corresponding region in the full-length 5 S rRNA.     

Structural organization of ribosomal RNAs from Novikoff hepatoma. II. Characterization of possible binding sites of 5 S rRNA and 5.8 S rRNA to 28 S rRNA

Interrelationships among 5 S, 5.8 S, and 28 S rRNA were probed by methods employed in the accompanying report (Choi, Y. C. (1985) J. Biol. Chem. 260, 12769-12772). Two complexes were isolated from 20 S ribonucleoprotein (RNP) fraction and 60 S subunit. The 20 S RNP fraction was found to contain the 3'-340 nucleotide fragment (domain VII) in association with 5 S rRNA. The 60 S subunit contained a stable complex consisting of the 5'-upstream portion (4220-4462, domain VI and VII), the 3'-downstream portion (4463-4802, domain VII) of 3'-583 nucleotides fragment, and 5.8 S rRNA. By computer analysis and hybridization, the 5'-upstream portion was found to contain the 5.8 S rRNA contact site. By affinity chromatography, the 3'-downstream portion was found to contain the 5 S rRNA association site. Furthermore, by comparison with the secondary structure of 28 S rRNA proposed by Hadjiolov et al. (Hadjiolov, A. A., Georgiev, O. I., Nosikov, V. V., and Yavachev, L. P. (1984) Nucleic Acids Res. 12, 3677-3693), it was found that domain VII is capable of binding 5.8 S rRNA and 5 S rRNA juxtaposed to each other. Accordingly, a model was proposed to indicate that a possible contact site for 5.8 S rRNA is within the region surrounding the alpha-sarcin site (4333-4350) and is a possible association site of 5 S rRNA within the 3'-downstream portion (4463-4802) of the 3'-583 nucleotide fragment (4220-4802).     

The participation of 5S rRNA in the co-translational formation of a eukaryotic 5S ribonucleoprotein complex

The eukaryotic ribosomal 5S RNA–protein complex (5S rRNP) is formed by a co-translational event that requires 5S rRNA binding to the nascent peptide chain of eukaryotic ribosomal protein L5. Binding between 5S rRNA and the nascent chain is specific: neither the 5S rRNA nor the nascent chain of L5 protein can be substituted by other RNAs or other ribosomal proteins. The region responsible for binding 5S rRNA is located at positions 35–50 with amino acid sequence RLVIQDIKNKYNTPKYRM. Eukaryotic 5S rRNA binds a nascent chain having this sequence, but such binding is not substantive enough to form a 5S-associated RNP complex, suggesting that 5S rRNA binding to the nascent chain is amino acid sequence dependent and that formation of the 5S rRNP complex is L5 protein specific. Microinjection of 5S rRNP complex into the cytoplasm of Xenopus oocytes results in both an increase in the initial rate and also in the extent of net nuclear import of L5. This suggests that the 5S rRNP complex enhances nuclear transport of L5. We propose that 5S rRNA plays a chaperone-like role in folding of the nascent chain of L5 and directs L5 into a 5S rRNP complex for nuclear entry.     

Localisation of part of the binding sites of 30S ribosomal proteins S4 and S20 in a small uninterrupted fragment of 16S RNA.

Analyses of the T1 ribonuclease-alkaline phosphatase fingerprint of a continuous fragment of the 16S rRNA, 170-230 nucleotides long, isolated from the products of autodigestion of 30S ribosome subunits show that it contains a sequence near the 5'-phosphate terminus of intact 16S rRNA and corresponds to segment H'-M of this molecule as defined by Ehresmann et al [29]. Incubation of this fragment with total 30S ribosomal proteins under reconstitution conditions leads to the formation of a complex containing proteins S4, S20, and one or both of proteins S16 and S17. The stoichiometry of these proteins in the complex is discussed.     

Primary structure of the 5'-region of Yersinia pestis 16S rRNA]

The sequence of 5'-region (16-296 n.) of 16S rRNA of plague agent (Yersinia pestis) was determined after sequencing of cloned cDNA fragments complementary to this region. When compared to the same region of 16S rRNA of Escherichia coli and Proteus vulgaris this region revealed 91.8% and 87.2% of homology, respectively. The sequences specific for Y. pestis 16S rRNA were localized and their secondary structure position was discussed.     

Probing the structure of 16 S ribosomal RNA from Bacillus brevis

A majority (approximately 89%) of the nucleotide sequence of Bacillus brevis 16 S rRNA has been determined by a combination of RNA sequencing methods. Several experimental approaches have been used to probe its structure, including (a) partial RNase digestion of 30 S ribosomal subunits, followed by two-dimensional native/denatured gel electrophoresis, in which base-paired fragments were directly identified; (b) identification of positions susceptible to cleavage by RNase A and RNase T1 in 30 S subunits; (c) sites of attack by cobra venom RNase on naked 16 S rRNA; and (d) nucleotides susceptible to attack by bisulfite in 16 S rRNA. These data are discussed with respect to a secondary structure model for B. brevis 16 S rRNA derived by comparative sequence analysis.     

The nucleotide sequence of 5S ribosomal RNA from Schizosaccharomyces pombe

The nucleotide sequence of 5S rRNA from the fission yeast, S. pombe, has been established by post labeling procedures combined with cataloging RNase T1- and A-oligonucleotides derived from unlabeled 5S rRNA. The sequence consists of 119 nucleotides without a modified base and shows more dissimilarities (at 38 positions) from that of S. cerevisiae than from that of humans (at 33 positions).     

Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences

In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization.     

Identification of ten additional nucleotides in the primary structure of yeast 18S rRNA

The ten-nucleotide-long sequence have been omitted while sequencing the 18S rRNA gene from yeast Saccharomyces cerevisiae [Rubtsov et al., Nucl. Acids Res. 8 (1980) 5779-5794]. This GAAGAUGAUC sequence and some other minor corrections are reintroduced into the yeast 18S rRNA primary structure.     

On the interaction of ribosomal protein L5 with 5S rRNA

Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel beta-sheet and four alpha-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692-701, 20011. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel beta-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at beta-strands and loop structures in BstL5. The mutation of Lys33 at the beta 1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the beta2-strand, the alpha3-beta4 loop, and the beta4-beta5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the beta2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the beta-sheet structure, and Phe77 at the beta2-beta3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the beta-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.     

Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA.

Eukaryotic 5S rRNA hybridizes specifically with 18S rRNA in vitro to form a stable intermolecular RNA:RNA hybrid. We have used 5S rRNA/18S rRNA fragment hybridization studies coupled with ribonuclease digestion and primer extension/chain termination analysis of 5S rRNA:18S rRNA hybrids to more completely map those mouse 5S rRNA and 18S rRNA sequences responsible for duplex formation. Fragment hybridization analysis has defined a 5'-terminal region of 5S rRNA (nucleotides 6-27) which base-pairs with two independent sequences in 18S rRNA designated Regions 1 (nucleotides 1157-1180) and 2 (nucleotides 1324-1339). Ribonuclease digestion of isolated 5S rRNA:18S rRNA hybrids with both single-strand- and double-strand-specific nucleases supports the involvement of this 5'-terminal 5S rRNA sequence in 18S rRNA hybridization. Primer extension/chain termination analysis of isolated 5S rRNA:18S rRNA hybrids confirms the base-pairing of 5S rRNA to the designated Regions 1 and 2 of 18S rRNA. Using these results, 5S rRNA:18S rRNA intermolecular hybrid structures are proposed. Comparative sequence analysis revealed the conservation of these hybrid structures in higher eukaryotes and the same but smaller core hybrid structures in lower eukaryotes and prokaryotes. This suggests that the 5S rRNA:16S/18S rRNA hybrids have been conserved in evolution for ribosome function.     

18S ribosomal RNA and tetrapod phylogeny

Previous phylogenetic analyses of tetrapod 18S ribosomal RNA (rRNA) sequences support the grouping of birds with mammals, whereas other molecular data, and morphological and paleontological data favor the grouping of birds with crocodiles. The 18S rRNA gene has consequently been considered odd, serving as "definitive evidence of different genes providing significantly different estimates of phylogeny in higher organisms" (p. 156; Huelsenbeck et al., 1996, Trends Ecol. Evol. 11:152-158). Our research indicates that the previous discrepancy of phylogenetic results between the 18S rRNA gene and other genes is caused mainly by (1) the misalignment of the sequences, (2) the inappropriate use of the frequency parameters, and (3) poor sequence quality. When the sequences are aligned with the aide of the secondary structure of the 18S rRNA molecule and when the frequency parameters are estimated either from all sites or from the variable domains where substitutions have occurred, the 18S rRNA sequences no longer support the grouping of the avian species with the mammalian species.