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).     

Prediction of the recognition sites on 16S and 23S rRNAs from E. coli for the formation of 16S-23S rRNA complex

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E. coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891-899 and 1195-1203 on 16S rRNA along with the corresponding complementary sites 1904-1912 and 760-768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630-638 on 16S rRNA with its complementary site 2031-2039 on 23S rRNA cannot be ruled out.     

Prediction of the Recognition Sites on 16S and 23S rRNAs from E.coli for the Formation of 16S-23S rRNA Complex

Abstract

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E.coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891–899 and 1195–1203 on 16S rRNA along with the corresponding complementary sites 1904–1912 and 760–768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630–638 on 16S rRNA with its complementary site 2031–2039 on 23S rRNA cannot be ruled out.     

Functional Escherichia coli 23S rRNAs containing processed and unprocessed intervening sequences from Salmonella typhimurium.

We have introduced the intervening sequence (IVS) from 23S rRNA of the rrnD operon of Salmonella typhimurium into the equivalent position of Escherichia coli 23S rRNA. Salmonella typhimurium 23S rRNA is fragmented due to the RNase III-dependent removal of the approximately 100 nt stem-loop structure that comprises the IVS. In this study, we have found that insertion of the S. typhimurium IVS into E. coli 23S rRNA causes fragmentation of the RNA but does not affect ribosome function. Cells expressing the fragmented 23S rRNA exhibited wild-type growth rates. Fragmented RNA was found in the actively translating polysome pool and did not alter the sedimentation profile of ribosomal subunits, 70S ribosomes or polysomes. Finally, hybrid 23S rRNA carrying the A2058G mutation conferred high level erythromycin resistance indistinguishable from that of intact 23S rRNA carrying this mutation. These observations indicate that the presence of this IVS and its removal are phenotypically silent. As observed in an RNase III-deficient strain, processing of the IVS was not required for the production of functional ribosomes.     

Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2'-O-methylations in 16S and 23S rRNAs

The cyclic peptide antibiotics capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Loss of these previously unidentified rRNA methylations confers resistance to capreomycin and viomycin. Many bacterial genera including enterobacteria lack a tlyA gene and the ensuing methylations and are less susceptible than mycobacteria to capreomycin and viomycin. We show that expression of recombinant tlyA in Escherichia coli markedly increases susceptibility to these drugs. When the ribosomal subunits associate during translation, the two tlyA-encoded methylations are brought into close proximity at interbridge B2a. The location of these methylations indicates the binding site and inhibitory mechanism of capreomycin and viomycin at the ribosome subunit interface.     

Sequences of the 5S rRNAs of the thermo-acidophilic archaebacterium Sulfolobus solfataricus (Caldariella acidophila) and the thermophilic eubacteria Bacillus acidocaldarius and Thermus aquaticus.

We have determined the nucleotide sequences of the 5 S rRNAs of three thermophilic bacteria: the archaebacterium Sulfolobus solfataricus, also named Caldariella acidophila, and the eubacteria Bacillus acidocaldarius and Thermus aquaticus. A 5 S RNA sequence for the latter species had already been published, but it looked suspect on the basis of its alignment with other 5 S RNA sequences and its base-pairing pattern. The corrected sequence aligns much better and fits in the universal five helix secondary structure model, as do the sequences for the two other examined species. The sequence found for Sulfolobus solfataricus is identical to that determined by others for Sulfolobus acidocaldarius. The secondary structure of its 5 S RNA shows a number of exceptional features which distinguish it not only from eubacterial and eukaryotic 5 S RNAs, but also from the limited number of archaebacterial 5 S RNA structures hitherto published. The free energy change of secondary structure formation is large in the three examined 5 S RNAs.     

Comparative analysis of 16S rRNA and amoA genes from archaea selected with organic and inorganic amendments in enrichment culture

We took advantage of a plant-root enrichment culture system to characterize mesophilic soil archaea selected through the use of organic and inorganic amendments. Comparative analysis of 16S rRNA and amoA genes indicated that specific archaeal clades were selected under different conditions. Three amoA sequence clades were identified, while for a fourth group, identified by 16S rRNA gene analysis alone and referred to as the "root" clade, we detected no corresponding amoA gene. The amoA-containing archaea were present in media with either organic or inorganic amendments, whereas archaea representing the root clade were present only when organic amendment was used. Analysis of amoA gene abundance and expression, together with nitrification-coupled growth assays, indicated potential growth by autotrophic ammonia oxidation for members of two group 1.1b clades. Increased abundance of one of these clades, however, also occurred upon the addition of organic amendment. Finally, although amoA-containing group 1.1a archaea were present in enrichments, we detected neither expression of amoA genes nor evidence for nitrification-coupled growth of these organisms. These data support a model of a diverse metabolic community in mesophilic soil archaea that is just beginning to be characterized.     

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.     

Post-translational modifications in the active site region of methyl-coenzyme M reductase from methanogenic and methanotrophic archaea

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. Isoenzyme I from Methanothermobacter marburgensiswas shown to contain a thioxo peptide bond and four methylated amino acids in the active site region. We report here that MCRs from all methanogens investigated contain the thioxo peptide bond, but that the enzymes differ in their post-translational methylations. The MS analysis included MCR I and MCR II from Methanothermobacter marburgensis, MCR I from Methanocaldococcus jannaschii and Methanoculleus thermophilus, and MCR from Methanococcus voltae, Methanopyrus kandleri and Methanosarcina barkeri. Two MCRs isolated from Black Sea mats containing mainly methanotrophic archaea of the ANME-1 cluster were also analyzed.     

Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by their interactions with mutant 23S rRNAs.

The ribosomal protein L11 binds to the region of 23S rRNA associated with the GTPase-dependent steps of protein synthesis. Nucleotides 1054-1107 within this region of the Escherichia coli 23S rRNA gene were mutagenized with bisulphite. Twenty point mutations (G-->A and C-->T transitions) and numerous multiple mutations were generated. Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably alter the phenotype, with effects ranging from a slight growth rate reduction to lack of viability. Temperature sensitivity is conferred by 1071G-->A and 1092C-->U substitutions. These effects are relieved by point mutations at other sites, indicating functional interconnections within the higher order structure of this 23S rRNA region. Several mutations prevent direct binding of r-protein L11 to 23S rRNA in vitro. These mutations are mainly in a short irregular stem (1087-1102) and within a hairpin loop (1068-1072), where the protein probably makes nucleotide contacts. Some of these mutations also interfere with binding of the r-protein complex L10.(L12)4 to an adjacent site on the rRNA. When added together to rRNA, proteins L10.(L12)4 and L11 bind cooperatively to overcome the effects of mutations at 1091 and 1099. The proteins also stimulate each others binding to rRNA mutated at 1087 or 1092, although in these cases binding remains clearly substoichiometric. Surprisingly, none of the mutations prevents incorporation of L11 into ribosomes in vivo, indicating that other, as yet unidentified, factors are involved in the cooperative assembly process.     

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 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.     

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.     

An extension of the graph theoretical approach to predict the secondary structure of large RNAs: the complex of 16S and 23S rRNAs from E.coli as a case study

An algorithm using the graph theoretical approach to predictsecondary structures of large nucleic acids is discussed. Reliabilityof prediction can be improved by incorporating available experimentaldata and sequence homology information. As a case study, thisalgorithm is applied to predict the secondary structure of the16S–23S rRNA complex from E. coli. It was found that severalstructures of the complex can coexist. The computer programdeveloped to predict the secondary structure of large RNAs canbe run on IBM PC/AT compatible systems. Received on November 20, 1988; accepted on April 15, 1989     

Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes. Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea

Synthesis of the nonbilayer-prone alpha-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the lipid surface charge density in the membrane of Acholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC ) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX(7)E catalytic motif of the CAZy family 4 of alpha-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity for these proteins. However, alMGS and spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface binding and activity is proposed for this regulatory enzyme.     

Structural analyses of plastid-derived 16S rRNAs in holoparasitic angiosperms

Higher-order structures have been constructed for plastid-encoded small-subunit (SSU, 16S), rRNAs from representatives of seven nonphotosynthetic holoparasitic angiosperm families: Apodanthaceae, Cynomoriaceae, Cytinaceae, Balanophoraceae, Hydnoraceae, Mitrastemonaceae, and Rafflesiaceae. Whereas most pairwise comparisons among angiosperms differ by 2–3% in substitutions, the 16S rRNAs of the holoparasites show an increasingly greater number of mutations: Cynomorium (7.3%), Cytinus (8.0%), Bdallophyton (12.7%), Mitrastema (14.9%), Hydnora (19.4%), Pilostyles (30.4%) and Corynaea (35.9%). Despite this high level of sequence variation, SSU structures constructed for all species except Pilostyles possess the typical complement of 50 helices (that contain numerous compensatory mutations) thereby providing indirect evidence supporting their functionality. Pilostyles, likely with the most unusual plastid 16S rRNA yet documented, lacks four major helices and contains lengthy insertions for four others. Sequences of products generated via RT-PCR show that these structural modifications are present on a mature (transcribed) rRNA. The trend toward increasing numbers of base substitutions in the holoparasites is accompanied by a marked increase in AA+U content of the rRNA. This A/T drift phenomenon of rDNA is especially apparent in Corynaea whose SSU rDNA sequence is 72% A+T. A comparison of Cytinus to tobacco showed that substitution rates appear to be dependent upon the composition of neighboring bases. Transversions represented 26% of the mutations when flanking bases were G or C whereas transversions increased to 36% when the flanking bases were A to T. The underlying molecular mechanism associated with these high substitution rates is presently unknown, however, relaxation of selection pressure on ribosome function resulting in altered DNA replication and/or repair systems may be involved.