The secondary structure of the protein L1 binding region of ribosomal 23S RNA. Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA.

An heterologous complex was formed between E. coli protein L1 and P. vulgaris 23S RNA. We determined the primary structure of the RNA region which remained associated with protein L1 after RNase digestion of this complex. We also identified the loci of this RNA region which are highly susceptible to T1, S1 and Naja oxiana nuclease digestions respectively. By comparison of these results with those previously obtained with the homologous regions of E. coli and B. stearothermophilus 23S RNAs, we postulate a general structure for the protein L1 binding region of bacterial 23S RNA. Both mouse and human mit 16S rRNAs and Xenopus laevis and Tetrahymena 28S rRNAs contain a sequence similar to the E. coli 23s RNS region preceding the L1 binding site. The region of mit 16S rRNA which follows this sequence has a potential secondary structure bearing common features with the L1-associated region of bacterial 23S rRNA. The 5'-end region of the L11 mRNA also has several sequence potential secondary structures displaying striking homologies with the protein L1 binding region of 23S rRNA and this probably explains how protein L1 functions as a translational repressor. One of the L11 mRNA putative structures bears the features common to both the L1-associated region of bacterial 23S rRNA and the corresponding region of mit 16S rRNA.     

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.     

Electron microscopic mapping of secondary structures in bacterial 16S and 23S ribosomal ribonucleic acid and 30S precursor ribosomal ribonucleic acid

Electron microscopy revealed reproducible secondary structure patterns within partially denatured 16S and 23S ribosomal ribonucleic acid (rRNA) from Escherichia coli. When prepared with 50% formamide-100 mM ammonium acetate, 16S rRNA included two small hairpins that appeared in over 50% of all molecules. Three open loops were observed with frequencies of less than 25%. In contrast, 23S rRNA included a terminal open loop and two additional large structures in over 75% of all molecules. These secondary structure patterns were conserved in the 16S and 23S rRNA from Pseudomonas aeruginosa. The secondary structure of the 30S precursor rRNA from the ribonclease III-deficient E. coli mutant AB105 was mapped after partial denaturation in 70% formamide-100 mM ammonium acetate. Two large open loops were superimposed on the 16S and 23S rRNA secondary structure patterns. These loops were the most frequent structures found on the precursor, and their stems coincided with ribonuclease III cleavage sites. A tentative 5'-3 orientation was determined for the secondary structure patterns of 16S and 23S rRNA from their relative locations within 30S precursor rRNA. The relation of secondary structure to ribosomal protein binding and ribonuclease III cleavage is discussed.     

Prediction of secondary structures of 16S and 23S rRNA fromEscherichia coli

Small and large subunits ofEscherichia coli ribosome have three different rRNAs, the sequences of which are known. However, attempts by three groups to predict secondary structures of 16S and 23S rRNAs have certain common limitations namely, these structures are predicted assuming no interactions among various domains of the molecule and only 40% residues are involved in base pairing as against the experimental observation of 60 % residues in base paired state. Recent experimental studies have shown that there is a specific interaction between naked 16S and 23S rRNA molecules. This is significant because we have observed that the regions (oligonucleotides of length 9–10 residues), in 16S rRNA which are complementary to those in 23S rRNA do not have internal complementary sequences. Therefore, we have developed a simple graph theoretical approach to predict secondary structures of 16S and 23S rRNAs. Our method for model building not only uses complete sequence of 16S or 23S rRNA molecule along with other experimental observations but also takes into account the observation that specific recognition is possible through the complementary sequences between 16S and 23S rRNA molecules and, therefore, these parts of the molecules are not used for internal base pairing. The method used to predict secondary structures is discussed. A typical secondary structure of the complex between 16S and 23S rRNA molecules, obtained using our method, is presented and compared Briefly with earlier model Building studies.     

A comparison of the solution structures and conformational properties of the somatic and oocyte 5S rRNAs of Xenopus laevis

The secondary and tertiary structures of Xenopus oocyte and somatic 5S rRNAs were investigated using chemical and enzymatic probes. The accessibility of both RNAs towards single-strand specific nucleases (T1, T2, A and S1) and a helix-specific ribonuclease from cobra venom (RNase V1) was determined. The reactivity of nucleobase N7, N3 and N1 positions towards chemical probes was investigated under native (5 mM MgCl2, 100 mM KCl, 20 degrees C) and semi-denaturing (1 mM EDTA, 20 degrees C) conditions. Ethylnitrosourea was used to identify phosphates not reactive towards alkylation under native conditions. The results obtained confirm the presence of the five helical stems predicted by the consensus secondary structure model of 5S rRNA. The chemical reactivity data indicate that loops C and D are involved in a number of tertiary interactions, and loop E folds into an unusual secondary structure. A comparison of the data obtained for the two types of Xenopus 5S rRNA indicates that the conformations of the oocyte and somatic 5S rRNAs are very similar. However, the data obtained with nucleases under native conditions, and chemical probes under semi-denaturing conditions, reveal that helices III and IV in the somatic 5S rRNA are less stable than the same structures in oocyte 5S rRNA. Using chimeric 5S rRNAs, it was possible to demonstrate that the relative resistance of oocyte 5S rRNA to partial denaturation in 4 M urea is conferred by the five oocyte-specific nucleotide substitutions in loop B/helix III. In contrast, the superior stability of oocyte 5S rRNA in the presence of EDTA is related to a single C substitution at position 79.     

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.     

The 3''-terminal region of bacterial 23S ribosomal RNA: structure and homology with the 3''-terminal region of eukaryotic 28S rRNA and with chloroplast 4.5s rRNA.

The sequence of the 110 nucleotide fragment located at the 3'-end of E.coli, P.vulgaris and A.punctata 23S rRNAs has been determined. The homology between the E.coli and P.vulgaris fragments is 90%, whereas that between the E.coli and A.punctate fragments is only 60%. The three rRNA fragments have sequences compatible with a secondary structure consisting of two hairpins. Using chemical and enzymatic methods recently developed for the study of the secondary structure of RNA, we demonstrated that one of these hairpins and part of the other are actually present in the three 3'-terminal fragments in solution. This supports the existence of these two hairpins in the intact molecule. Indeed, results obtained upon limited digestion of intact 23S RNA with T1 RNase were in good agreement with the existence of these two hairpins. We observed that the primary structures of the 3'-terminal regions of yeast 26S rRNA and X.laevis 28S rRNA are both compatible with a secondary structure similar to that found at the 3'-end of bacterial 23S rRNAs. Furthermore, both tobacco and wheat chloroplast 4.5S rRNAs can also be folded in a similar way as the 3'-terminal region of bacterial 23S rRNA, the 3'-end of chloroplast 4.5S rRNAs being complementary to the 5'-end of chloroplast 23S rRNA. This strongly reinforces the hypothesis that chloroplast 4.5S rRNA originates from the 3'-end of bacterial 23S rRNA and suggests that this rRNA may be base-paired with the 5'-end of chloroplast 23S rRNA. Invariant oligonucleotides are present at identical positions in the homologous secondary structures of E.coli 23S, yeast 26S, X.laevis 28S and wheat and tobacco 4.5S rRNAs. Surprisingly, the sequences of these oligonucleotides are not all conserved in the 3'-terminal regions of A.punctata or even P.vulgaris 23S rRNAs. Results obtained upon mild methylation of E.coli 50S subunits with dimethylsulfate strongly suggest that these invariant oligonucleotides are involved in RNA tertiary structure or in RNA-protein interactions.     

Electron microscopy of secondary structure in partially denatured precursor and mature Escherichia coli 16 S and 23 S rRNA

The secondary structure of 16 S and 23 s rRNA sequences in 30 S preribosomal RNA of Escherichia coli was analyzed by electron microscopy after partial denaturation and compared to mature 16 S and 23 S rRNA examined under the same conditions. The sequences in the pre-rRNA notably lack the specific loops that dominate the 5'-terminal regions of mature 16 S and 23 S rRNA. In other respects, the sizes and locations of loops in the 23 S rRNA sequence are qualitatively very similar in mature and pre-rRNA. Eleven of 12 loops outside of the 5'-terminal domain correspond, with the most frequent features in the 3'-half of the molecule. In contrast, the sizes and locations of loops in the 16 S rRNA sequence differ between precursor and mature forms. In the pre-rRNA, instead of the 370-nucleotide 5'-terminal loop of mature rRNA, some 1000-nucleotide terminal loops are observed. The pre-rRNA also shows a frequent 610-nucleotide central loop and a large 1240-nucleotide loop not seen in the mature rRNA. Also, in the 3'-region of the sequence, the largest loops in pre-rRNA are 120 nucleotides shorter than in mature rRNA. We suggest that the structure of pre-rRNA may promote some alternate conformational features, and that these could be important during ribosome formation or function.     

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.     

The lonepair triloop: a new motif in RNA structure

The lonepair triloop (LPTL) is an RNA structural motif that contains a single ("lone") base-pair capped by a hairpin loop containing three nucleotides. The two nucleotides immediately outside of this motif (5' and 3' to the lonepair) are not base-paired to one another, restricting the length of this helix to a single base-pair. Four examples of this motif, along with three tentative examples, were initially identified in the 16S and 23S rRNAs with covariation analysis. An evaluation of the recently determined crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits revealed the authenticity for all of these proposed interactions and identified 16 more LPTLs in the 5S, 16S and 23S rRNAs. This motif is found in the T loop in the tRNA crystal structures. The lonepairs are positioned, in nearly all examples, immediately 3' to a regular secondary structure helix and are stabilized by coaxial stacking onto this flanking helix. In all but two cases, the nucleotides in the triloop are involved in a tertiary interaction with another section of the rRNA, establishing an overall three-dimensional function for this motif. Of these 24 examples, 14 occur in multi-stem loops, seven in hairpin loops and three in internal loops. While the most common lonepair, U:A, occurs in ten of the 24 LPTLs, the remaining 14 LPTLs contain seven different base-pair types. Only a few of these lonepairs adopt the standard Watson-Crick base-pair conformations, while the majority of the base-pairs have non-standard conformations. While the general three-dimensional conformation is similar for all examples of this motif, characteristic differences lead to several subtypes present in different structural environments. At least one triloop nucleotide in 22 of the 24 LPTLs in the rRNAs and tRNAs forms a tertiary interaction with another part of the RNA. When a LPTL containing the GNR or UYR triloop sequence forms a tertiary interaction with the first (and second) triloop nucleotide, it recruits a fourth nucleotide to mediate stacking and mimic the tetraloop conformation. Approximately half of the LPTL motifs are in close association with proteins. The majority of these LPTLs are positioned at sites in rRNAs that are conserved in the three phylogenetic domains; a few of these occur in regions of the rRNA associated with ribosomal function, including the presumed site of peptidyl transferase activity in the 23S rRNA.     

Computer analysis of potential stem structures of rRNA operons in various procaryote genomes

The processing of 16S rRNA and 23S rRNA by RNase III in E.coli is known to involve stem structures formed by both ends of the rRNA. Indeed, complementary nucleotide sequences are usually found at both ends of 16S rRNA and 23S rRNA. However, whether or not this phenomenon exists in various other bacteria has not yet been adequately studied. We have conducted computer analyses of potential stem structures of rRNA operons in 12 bacterial and 3 archaeal genomes, and compared characteristics of the stem structures among these species. We systematically computed free energy values by exhaustively 'annealing' sequences around the 5' end and sequences around the 3' end of both 16S rRNA and 23S rRNA genes, in order to predict potential stem structures.The results suggest that rRNAs in most species form stem structures at both ends. Some species, such as A.aeolicus, seem to form unusually stable stem structures. On the other hand, some rRNAs, such as rRNAs of D.radiodurans, seem not to form solid stem structures. This suggests that rRNA processing in those species must employ a reliable targeting mechanism other than recognizing stem structures by RNase III.     

Paramecium mitochondrial genes. II. Large subunit rRNA gene sequence and microevolution

Mature Paramecium mitochondrial large subunit rRNA consists of two stable segments: a 20 S segment described previously and a unique 283-base segment similar to 5.8 S rRNAs typically found in eucaryotic cytoplasmic RNA. pBR325 clones of both gene regions from both Paramecium primaurelia and Paramecium tetraurelia were sequenced and aligned. The gene segments lie adjacent to each other very near the replicative terminal end of the linear Paramecium mitochondrial genome and are transcribed from a common 23 S precursor. The precise gene ends were determined using nuclease S1 protection; the large subunit rRNA gene complex (consisting of "5.8 S-like" rRNA, a 19-26-base excised region, and 20 S rRNA) spans about 2654 base pairs. The gene complex is preceded by a 15-base poly(T) tract and terminates randomly within a 20-base A + T-rich segment immediately preceding the tRNATyr gene. The sequences from the two species were 4% divergent, the changes consisting of 59% transitions, 38% transversions, and 3% insertions or deletions. The sequences were aligned with Escherichia coli 23 S rRNA, and a secondary structure model is presented for the entire molecule based on structures proposed for E. coli 23 S rRNA.     

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii: Evolutionary and functional implications

The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoë, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.     

Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21.

The number of rRNA genes in Streptomyces lividans was examined by Southern hybridization. Randomly labeled 23 and 16S rRNAs were hybridized with BamHI, BglII, PstI, SalI, or XhoI digests of S. lividans TK21 DNA. BamHi, BglII, SalI and XhoI digests yielded six radioactive bands each for the 23 and 16S rRNAs, whereas PstI digests gave one band for the 23S rRNA and one high-intensity band and six low-density bands for the 16S rRNA. The 7.4-kilobase-pair BamHI fragment containing one of the rRNA gene clusters was cloned into plasmid pBR322. The hybrid plasmid, pSLTK1, was characterized by physical mapping, Southern hybridization, and electron microscopic analysis of the R loops formed between pSLTK1 and the 23 and 16S rRNAs. There were at least six rRNA genes in S. lividans TK21. The 16 and 23S rRNA genes were estimated to be about 1.40 and 3.17 kilobase pairs, respectively. The genes for the rRNAs were aligned in the sequence 16S-23S-5S. tRNA genes were not found in the spacer region or in the context of the rRNA genes. The G + C content of the spacer region was calculated to be approximately 58%, in contrast to 73% for the chromosome as a whole.     

Higher plant 5S rRNAs share common secondary and tertiary structure. A new three domains model

A new model of secondary and tertiary structure of higher plant 5S RNA is proposed. It consists of three helical domains: domain alpha includes stem I; domain beta contains stems II and III and loops B and C; domain gamma consists of stems IV and V and loops D and E. Except for, presumably, a canonical RNA-A like domain alpha, the two remaining domains apparently adopt a perturbed RNA-A structure due to irregularities within internal loops B and E and three bulges occurring in the model. Bending of RNA could bring loops B and E and/or C and D closer making tertiary interactions likely. The model differs from that suggested for eukaryotic 5S rRNA, by organization of domain gamma. Our model is based on the results of partial digestion obtained with single- and double-strand RNA specific nucleases. The proposed secondary structure is strongly supported by the observation that crude plant 5S rRNA contains abundant RNA, identified as domain gamma of 5S rRNA. Presumably it is excised from the 5S rRNA molecule by a specific nuclease present in lupin seeds. Experimental results were confirmed by computer-aided secondary structure prediction analysis of all higher plant 5S rRNAs. Differences observed between earlier proposed models and our proposition are discussed.     

A comparison of thermodynamic foldings with comparatively derived structures of 16S and 16S-like rRNAs.

To increase our understanding of the dynamics and complexities of the RNA folding process, and therewith to improve our ability to predict RNA secondary structure by computational means, we have examined the foldings of a large number of phylogenetically and structurally diverse 16S and 16S-like rRNAs and compared these results with their comparatively derived secondary structures. Our initial goals are to establish the range of prediction success for this class of rRNAs, and to begin comparing and contrasting the foldings of these RNAs. We focus here on structural features that are predicted with confidence as well as those that are poorly predicted. Whereas the large set of Archaeal and (eu)Bacterial 16S rRNAs all fold well (69% and 55% respectively), some as high as 80%, many Eucarya and mitochondrial 16S rRNAs are poorly predicted (approximately 30%), with a few of these predicted as low as 10-20%. In general, base pairs interacting over a short distance and, in particular, those closing hairpin loops, are predicted significantly better than long-range base pairs and those closing multistem loops and bulges. The prediction success of hairpin loops varies, however, with their size and context. Analysis of some of the RNAs that do not fold well suggests that the composition of some hairpin loops (e.g., tetraloops) and the higher frequency of noncanonical pairs in their comparatively derived structures might contribute to these lower success rates. Eucarya and mitochondrial rRNAs reveal further novel tetraloop motifs, URRG/A and CRRG, that interchange with known stable tetraloop in the procaryotes.