Functional studies of the 900 tetraloop capping helix 27 of 16S ribosomal RNA

The 900 tetraloop (positions 898-901) of Escherichia coli 16S rRNA caps helix 27, which is involved in a conformational switch crucial for the decoding function of the ribosome. This tetraloop forms a GNRA motif involved in intramolecular RNA-RNA interactions with its receptor in helix 24 of 16S rRNA. It is involved also in an intersubunit bridge, via an interaction with helix 67 in domain IV of 23S rRNA. Using a specialized ribosome system and an instant-evolution procedure, the four nucleotides of this loop were randomized and 15 functional mutants were selected in vivo. Positions 899 and 900, responsible for most of the tetraloop/receptor interactions, were found to be the most critical for ribosome activity. Functional studies showed that mutations in the 900 tetraloop impair subunit association and decrease translational fidelity. Computer modeling of the mutations allows correlation of the effect of mutations with perturbations of the tetraloop/receptor interactions.     

Functional Study of the Residue C899 in the 900 Tetraloop of Escherichia coli Small Subunit Ribosomal RNA

A mutant ribosome bearing C899G in the 900 tetraloop of Escherichia coli 16S rRNA, one implicated in a conformational switch in the dynamic movements of the ribosome, showed defects in subunit association and 30S initiation complex formation. Our results explain the basis of the loss of protein synthesis ability caused by a perturbation of the 900 tetraloop.     

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA.     

Role of N-terminal helix in interaction of ribosomal protein S15 with 16S rRNA

The position and conformation of the N-terminal helix of free ribosomal protein S15 was earlier found to be modified under various conditions. This variability was supposed to provide the recognition by the protein of its specific site on 16S rRNA. To test this hypothesis, we substituted some amino acid residues in this helix and assessed effects of these substitutions on the affinity of the protein for 16S rRNA. The crystal structure of the complex of one of these mutants (Thr3Cys S15) with the 16S rRNA fragment was determined, and a computer model of the complex containing another mutant (Gln8Met S15) was designed. The available and new information was analyzed in detail, and the N-terminal helix was concluded to play no significant role in the specific binding of the S15 protein to its target on 16S rRNA.     

16S rRNA序列分析法在医学微生物鉴定中的应用

１６Ｓ ｒＲＮＡ序列分析作为微生物系统分类的主要依据已得到了广泛认同,随着微生物核糖体数据库的日益完善,该技术成为细菌分类和鉴定的一个有力工具。本文概述了 １６５ ｒＲＮＡ序列分析法的技术步骤以及该技术在医学微生物研究中的应用,总结了目前文献报导的各种致病微生物种属特异性 １６５ ｒＲＮＡ引物和探针序列,同时分析了该技术在应用中存在的一些问题。     

Assembly of the 5' and 3' minor domains of 16S ribosomal RNA as monitored by tethered probing from ribosomal protein S20

The ribosomal protein (r-protein) S20 is a primary binding protein. As such, it interacts directly and independently with the 5′ domain as well as the 3′ minor domain of 16S ribosomal RNA (rRNA) in minimal particles and the fully assembled 30S subunit. The interactions observed between r-protein S20 and the 5′ domain of 16S rRNA are quite extensive, while those between r-protein S20 and the 3′ minor domain are significantly more limited. In this study, directed hydroxyl radical probing mediated by Fe(II)-derivatized S20 proteins was used to monitor the folding of 16S rRNA during r-protein association and 30S subunit assembly. An analysis of the cleavage patterns in the minimal complexes [16S rRNA and Fe(II)-S20] and the fully assembled 30S subunit containing the same Fe(II)-derivatized proteins shows intriguing similarities and differences. These results suggest that the two domains, 5′ and 3′ minor, are organized relative to S20 at different stages of assembly. The 5′ domain acquires, in a less complex ribonucleoprotein particle than the 3′ minor domain, the same architecture as observed in mature subunits. These results are similar to what would be predicted of subunit assembly by the 5′-to-3′ direction assembly model.     

Thermodynamics of the pseudo‐knot in helix 18 of 16S ribosomal RNA

A fragment of E. coli 16S rRNA formed by nucleotides 500 to 545 is termed helix 18. Nucleotides 505‐507 and 524‐526 form a pseudo‐knot and its distortion affects ribosome function. Helix 18 isolated from the ribosome context is thus an interesting fragment to investigate the structural properties and folding of RNA with pseudo‐knots. With all‐atom molecular dynamics simulations, spectroscopic and gel electrophoresis experiments, we investigated thermodynamics of helix 18, with a focus on its pseudo‐knot. In solution studies at ambient conditions we observed dimerization of helix 18. We proposed that the loop, containing nucleotides forming the pseudo‐knot, interacts with another monomer of helix 18. The native dimer is difficult to break but introducing mutations in the pseudo‐knot indeed assured a monomeric form of helix 18. Molecular dynamics simulations at 310 K confirmed the stability of the pseudo‐knot but at elevated temperatures this pseudo‐knot was the first part of helix 18 to lose the hydrogen bond pattern. To further determine helix 18 stability, we analyzed the interactions of helix 18 with short oligomers complementary to a nucleotide stretch containing the pseudo‐knot. The formation of higher‐order structures by helix 18 impacts hybridization efficiency of peptide nucleic acid and 2'‐O methyl RNA oligomers.     

A novel mutation 3090 G>A of the mitochondrial 16S ribosomal RNA associated with myopathy

We describe a young woman who presented with a progressive myopathy since the age of 9. Spectrophotometric analysis of the respiratory chain in muscle tissue revealed combined and profound complex I, III, II+III, and IV deficiency ranging from 60% to 95% associated with morphological and histochemical abnormalities of the muscle. An exhaustive screening of mitochondrial transfer and ribosomal RNAs showed a novel G>A substitution at nucleotide position 3090 which was detected only in urine sediment and muscle of the patient and was not found in her mother's blood cells and urine sample. We suggest that this novel de novo mutation in the 16S ribosomal RNA, a nucleotide which is highly conserved in different species, would impair mitochondrial protein synthesis and would cause a severe myopathy.     

Site of functional interaction of release factor 1 with the ribosome

Ribosomal protein L11 consists of a C-terminal and an N-terminal domain. To determine the importance of each domain for interaction with release factor 1, which works specifically at the UAG termination codon, we constructed Escherichia coli strains lacking either the entire L11 protein or just the N-terminal portion. Strains lacking L11 exhibited UAG suppression, defective growth, and high-temperature lethality, phenotypes that were reversed by expression of L11 protein from a plasmid. Strains lacking only the N-terminal portion of L11 grew well at physiological temperatures and survived at high temperature, but they were defective in UAG-dependent termination. Our results show for the first time that it is precisely the N-terminal part of ribosomal protein L11 that is required for the functional interaction of release factor 1 with the ribosome in the cell.     

Dead-end evolution of the Cnidaria as deduced from 5S ribosomal RNA sequences

Using nucleotide sequences of 5S ribosomal RNAs from 2 hydrozoan jellyfishes, 3 scyphozoan jellyfishes and 2 sea anemones, a phylogenetic tree of Cnidaria has been constructed to elucidate the evolutionary relationships of radial and bilateral symmetries. The 3 classes of Cnidaria examined herein belong to one branch, which does not include other metazoan phyla such as the Platyhelminthes. The Hydrozoa (having radial symmetry without septa) and the Scyphozoa (having radial symmetry with septa) are more closely related to each other than to the Anthozoa (having bilateral symmetry with septa). In classical taxonomy, multicellular animals are considered to have evolved through organisms with radial symmetry (e.g., Cnidaria) to bilateral symmetry. Our results, however, indicate that the emergence of the Bilateria was earlier than that of the Radiata, suggesting (in opposition to Haeckel's view) that the radial symmetry of Cnidaria is an evolutionary dead end.     

Identification of probiotic lactobacilli used for animal feeds on the basis of 16S ribosomal RNA gene sequence

The use of probiotics such as Lactobacillus in animal feeds has gained popularity in recent years. In this study the 16S rRNA gene sequence of L. acidophilus in two commercial agents which have been used in animal feeds, LAB‐MOS and Ghenisson 22, was determined. Phylogenetic tree analysis revealed that the two agents, strain MNFLM01 in LAB‐MOS and strain GAL‐2 in Ghenisson 22, belonged to L. rhamnosus (a member of the L. casei group) and L. johnsonii (a member of the L. acidophilus group), respectively. Biochemical tests assigned the two as L. rhamnosus and ambiguously as L. acidophilus. The data suggest that 16S rRNA gene sequence analysis provides more accurate identification of Lactobacillus species than biochemical tests and would allow quality assurance of relevant commercial products. The 16S rRNA gene sequences of strains MNFLM01 and GAL‐2 determined in this study have been submitted to the DDBJ/EMBL/GenBank accession numbers under accession numbers AB288235 and AB295648, respectively.     

Phylogenetic positions of the aberrant branchiobdellidans and aphanoneurans within the Annelida as derived from 18S ribosomal RNA gene sequences

Different hypotheses have been proposed on the phylogenetic relationships of branchiobdellidans and aphanoneurans among the Annelida based on the anatomical and embryological characters. The 18S ribosomal RNA gene sequences have been analyzed from representatives of the three major taxa of the Annelida plus the branchiobdellidans and aphanoneurans to assess their phylogenetic relationships to each other. In this preliminary study, all of the phylogenetic analyses show the branchiobdellidans as a sister group to the leeches, rather than the oligochaetes. The position of the aphanoneurans is stable as an independent taxon that evolved after the polychaetes branched from the evolutionary stem, but before the ancestral oligochaetes emerged.     

Probing the stabilizing effects of modified nucleotides in the bacterial decoding region of 16S ribosomal RNA

1. Download : Download full-size image

     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Role of precursor sequences in the ordered maturation of E. coli 23S ribosomal RNA

The maturation of ribosomal RNAs (rRNAs) is an important but incompletely understood process required for rRNAs to become functional. In order to determine the enzymes responsible for initiating 3' end maturation of 23S rRNA in Escherichia coli, we analyzed a number of strains lacking different combinations of 3' to 5' exo-RNases. Through these analyses, we identified RNase PH as a key effector of 3' end maturation. Further analysis of the processing reaction revealed that the 23S rRNA precursor contains a CC dinucleotide sequence that prevents maturation from being performed by RNase T instead. Mutation of this dinucleotide resulted in a growth defect, suggesting a strategic significance for this RNase T stalling sequence to prevent premature processing by RNase T. To further explore the roles of RNase PH and RNase T in RNA processing, we identified a subset of transfer RNAs (tRNAs) that contain an RNase T stall sequence, and showed that RNase PH activity is particularly important to process these tRNAs. Overall, the results obtained point to a key role of RNase PH in 23S rRNA processing and to an interplay between this enzyme and RNase T in the processing of different species of RNA molecules in the cell.     

Structural dynamics of bacterial ribosomes. III. Quantitative conversion of vacant ribosome couples into an initiation complex with R17 RNA as messenger

The arrangement of the DNA sequences coding for the ribosomal 5.8 S RNA in the genome of Xenopus laevis has been studied. In Xenopus the 5.8 S cistrons, like the ribosomal 28 S and 18 S cistrons, are reiterated some 600-fold (Clarkson et al., 1973a). When banded in caesium chloride, the 5.8 S cistrons separate from somatic DNA of high molecular weight and band as a distinct satellite, indicating a clustered arrangement in the genome. The buoyant density of this satellite (1.723 g cm^?3) corresponds to that of the ribosomal DNA satellite.It has previously been shown that the ribosomal DNA sequences have been deleted from the genome of the anucleotide Xenopus mutant. Our findings, first that the anucleolate mutant does not synthesize 5.8 S RNA and second that somatic DNA from this mutant does not detectably hybridize with 5.8 S RNA, demonstrate that the 5.8 S cistronic complement has been similarly deleted. This finding supports our contention that 5.8 S sequences are clustered on chromosomal DNA and further suggests that they are located close to or within the rDNA complements in the nucleolus organizer region.Pre-hybridization to saturation with unlabelled 5.8 S RNA results in only a slight increase in the buoyant density of denatured 5.8 S coding sequences from low molecular weight DNA. Since a contiguous arrangement of the 5.8 S sequences would give rise to a much larger increase in density, it follows that, although clustered, the sequences must be intercalated within stretches of other DNA. By contrast, pre-hybridization of the somatic DNA with unlabelled 28 S or 18 S ribosomal RNAs results in large shifts in the buoyant density of the 5.8 S sequences. These shifts indicate that the 5.8 S sequences are closely linked to both 28 S and 18 S coding sequences.It is concluded that the 5.8 S cistrons are interspersed along the ribosomal DNA sense strand and that each is located together with a 28 S and an 18 S cistron in a ribosomal repeat unit. Estimates, obtained from the pre-hybridization experiments, of the separations between the 5.8 S and the 28 S and 18 S sequences, are combined in a model of the ribosomal repeat unit. In this model the 5.8 S cistron is located within the transcribed spacer which links the 28 S and 18 S coding sequences.