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.     

Heterogeneous rRNA molecules encoded by Streptomyces coelicolor M145 genome are all expressed and assembled into ribosomes

The Streptomyces coelicolor M145 genome harbors six copies of divergent rRNA operons that differ at ~0.2% and ~0.6% of the nucleotide positions in small subunit (SSU) and large subunit (LSU) rRNA genes, respectively. When these rRNA genes are expressed, a single cell may harbor three different kinds of SSU rRNA and five kinds of LSU rRNA. Primer extension analyses revealed that all of the heterogeneous rRNA molecules are expressed and assembled into ribosomes. This finding and the maintenance of the intragenomic variability of rRNA operons imply the existence of functional divergence of rRNA species in this developmentally complex microorganism.     

Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus.

We examined the sequence variability of the nontranscribed spacer (NTS) and internal-transcribed spacer (ITS1 and ITS2) domains of the rRNA locus of Perkinsus marinus from Maryland, Florida, and Louisiana. The sequence of P. marinus DNA including the 5S rRNA, NTS, small subunit (SSU) rRNA, ITSI, and ITS2 regions confirmed their contiguity in the rRNA locus and revealed differences at 28 positions with the SSU rRNA sequences published earlier. The 307-bp polymerase chain reaction (PCR)-amplified fragments from the NTS domain of the various P. marinus isolates revealed the presence of 2 distinct sequences, designated as types I and II, that differed at 6 defined nucleotide positions. Based on these differences, nested PCR and restriction enzyme digests were used to distinguish between the 2 types. Sequences of the ITS1 and ITS2 domains of samples from either NTS type I (n = 3) or type II (n = 3) showed no variation and were identical to published sequences. Frequencies of the P. marinus NTS sequence types I and II in infected oysters varied with the geographic origin of the samples. All Maryland samples examined (n = 19) corresponded to the NTS type I sequence, the type II was the most frequent in the Florida samples (n = 17), and both types were about equally represented in the Louisiana samples (n = 19), with both sequence types found in individual oyster specimens. Although it has been suggested that P. marinus is diploid, it remains to be determined if both NTS sequence types can be present in a single P. marinus trophozoite.     

Phylogenetic Diversity of Parabasalian Symbionts from Termites, Including the Phylogenetic Position of Pseudotrypanosoma and Trichonympha

ABSTRACT The phylogenetic diversity of parabasalian flagellates from termite hindguts has been examined by small subunit ribosomal RNA (rRNA) amplification and sequencing. Two species of particular interest, the giant trichomonad Pseudotrypanosoma giganteum and the hypermastigote Trichonympha magna, were isolated from the gut of Porotermes adamsoni by micropipetting. and the rRNA genes from these small populations amplified and sequenced. rRNA genes representing Hypermastigida and the Trichomonadida families Devescovinidae and Trichomonadidae. were also recovered by amplification from whole hindguts of three termites, P. adamsoni, Cryptotermes brevis , and Cryptotermes dudleyi. The parabasalian rRNA genes from C. brevis were found to comprise a unique and extremely heterogeneous lineage with no clear affinities to any known parabasalian rRNAs. In addition, one of the sequences isolated from P. Adamsoni was found to be similar to another uncharacterised rRNA gene from Reticulitermes flavipes. The phylogeny of all known parabasalian small subunit rRNAs was examined with these new sequences. We find many taxonomic groups to be supported by rRNA, but not all. We have found the root of parabasalia to be very difficult to discern accurately, but have nevertheless identified several possible positions.     

Divergent Histories of rDNA Group I Introns in the Lichen Family Physciaceae

The wide but sporadic distribution of group I introns in protists, plants, and fungi, as well as in eubacteria, likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA, which can support the insertion and/or retention of group I introns. In contrast, we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene. Reviewing Editor: Dr. W. Ford Doolittle     

Segments of 18S rRNA, located in the area of the mRNA-binding center of ribosomes from human placenta

The affinity labelling of human placenta 80S ribosomes by 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphoramide of hexauridylate has been studied. This mRNA analogue has normal coding properties because its binding to placenta ribosomes significantly increases in the presence of the cognate tRNA(Phe). Incubation of the mRNA analogue in the complex with ribosomes and Phe-tRNAPhe) leads to its covalent attachment exclusively to the small subunit (mainly to 18S rRNA). The reaction site has been shown by hybridisation experiments to be located within positions 975-1055 of 18S rRNA. The identified fragment is located in a highly conserved part of the small subunit rRNA domain II.     

The formation of a defective small subunit of the mitochondrial ribosomes in petite mutants of Saccharomyces cerevisiae

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in petite mutants of Saccharomyces cerevisiae which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes and/or one of the rRNA genes on the mtDNA. Petite strains which retain the 15-S rRNA gene can synthesize this rRNA species, but do not contain any detectable amounts of the small mitochondrial ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S (instead of 37 S) was observed. This ribonucleoparticle contained all the small ribosomal subunit proteins with the exception of the var1 and three to five other proteins, which indicates that the 30-S ribonucleoparticle is related to the small mitochondrial ribosomal subunit (37 S). Reconstitution experiments using the 30-S particle and the large mitochondrial ribosomal subunit from a wild-type yeast strain indicate that the 30-S particle is not active in translating the artificial message poly(U). The large mitochondrial ribosomal subunit was present in petite strains retaining the 21-S rRNA gene. The petite 54-S subunit is biologically active in the translation of poly(U) when reconstituted with the small subunit (37 S) from a wild-type strain. The above results indicate that mitochondrial protein synthetic activity is essential for the assembly of the mature small ribosomal subunit, but not for the large subunit. Since the var1 protein is the only mitochondrial translation product known to date to be associated with the mitochondrial ribosomes, the results suggest that this protein is essential for the assembly of the mature small subunit.     

Identification of a site on 18 S rRNA of human placenta ribosomes in the region of the mRNA binding center

The affinity labeling of human placenta 80 S ribosomes by 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide of hexauridylate was studied. This mRNA analog has normal coding properties because its binding to placenta ribosomes significantly increases in the presence of cognate tRNAPhe. Incubation of the mRNA analog in the complex with the ribosomes and Phe-tRNAPhe leads to its covalent attachment exclusively to the small subunit (mainly to 18 S rRNA). The site of the reaction has been identified by hybridization experiments to be located within positions 975 to 1055 of 18 S rRNA. The identified fragment is located in a highly conserved part of the small subunit rRNA domain II.     

Structural-functional topography of human ribosomes based on the data from crosslinking with mRNA analogs--oligoribonucleotide derivatives

Reviewed are data on the position of template codons with respect to 18S rRNA and certain proteins on human ribosome obtained using a set of mRNA analogs, oligoribonucleotide derivatives carrying alkylating or photoactivatable group at different positions. A comparison of data on template position on the human and Escherichia coli ribosomes has revealed both the similarity in the structure of the mRNA-binding site of bacterial and mammalian ribosomes and the peculiarities of the functioning of mammalian (in particular, human) ribosomes. The similarity manifests itself in that the template codons at the A, P, and E sites of bacterial and human ribosomes are surrounded by similar nucleotides (occupying similar positions in the conserved regions of secondary structure) of small subunit rRNA. The template forms a loop whose foot is in proximity to the 530 stem-loop conserved region of rRNA. The specific features of mammalian ribosomes appear to be associated with their lower conformational mobility as compared with bacterial ribosomes, owing to which their interaction with the template involves a lesser number of molecular contacts.     

Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20.

The 16S ribosomal RNA neighborhood of ribosomal protein S20 has been mapped, in both 30S subunits and 70S ribosomes, using directed hydroxyl radical probing. Cysteine residues were introduced at amino acid positions 14, 23, 49, and 57 of S20, and used for tethering 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA. In vitro reconstitution using Fe(II)-derivatized S20, together with the remaining small subunit ribosomal proteins and 16S ribosomal RNA (rRNA), yielded functional 30S subunits. Both 30S subunits and 70S ribosomes containing Fe(II)-S20 were purified and hydroxyl radicals were generated from the tethered Fe(II). Hydroxyl radical cleavage of the 16S rRNA backbone was monitored by primer extension. Different cleavage patterns in 16S rRNA were observed from Fe(II) tethered to each of the four positions, and these patterns were not significantly different in 30S and 70S ribosomes. Cleavage sites were mapped to positions 160-200, 320, and 340-350 in the 5' domain, and to positions 1427-1430 and 1439-1458 in the distal end of the penultimate stem of 16S rRNA, placing these regions near each other in three dimensions. These results are consistent with previous footprinting data that localized S20 near these 16S rRNA elements, providing evidence that S20, like S17, is located near the bottom of the 30S subunit.     

A partial phylogenetic analysis of the "flavobacter-bacteroides" phylum: basis for taxonomic restructuring

On the basis of small subunit rRNA sequence analyses five major subgroups within the flavobacteria-bacteroides phylum have been defined. These are tentatively designated the cytophaga subgroup (comprising largely Cytophaga species), the flavobacter subgroup (comprising the true flavobacteria and the polyphyletic genus Weeksella), the bacteroides subgroup (comprising the bacteroides and certain cytophaga-like bacteria), the sphingobacter subgroup (which contains the known sphingolipid-producing members of the phylum), and the saprospira subgroup (comprising particular species of Flexibacter, Flavobacterium, Haliscomenobacter, and, of course, the genus Saprospira). These groupings are given not only by evolutionary distance analysis, but can be defined and distinguished on the basis of a simple small subunit rRNA signatures.     

Structure of 5S rRNA within the Escherichia coli ribosome: iodine-induced cleavage patterns of phosphorothioate derivatives.

The protection patterns of 5S rRNA in solution, within the ribosomal 50S subunit, 70S ribosomes, and functional complexes, were assessed with the phosphorothioate method. About 20% of the analyzed positions (G9-G107) showed strong assembly defects: A phosphorothioate at one of these positions significantly impaired the incorporation of 5S rRNA into 50S particles. The reverse has also been observed: A phosphorothioate is preferred over a phosphate residue in the assembly process at a few positions. The results further demonstrate that 5S rRNA undergoes conformational changes during the assembly in the central protuberance of the 50S subunit and upon association with the small ribosomal subunit forming a 70S ribosome. In striking contrast, when the 70S ribosomes are once formed, the contact pattern of the 5S rRNA is the same in various functional states such as initiation-like complexes and pre- and posttranslocational states.     

Structural and Functional Topography of Human Ribosomes Deduced from Crosslinking with mRNA Analogs,Oligoribonucleotide Derivatives

Reviewed are data on the position of template codons with respect to 18S rRNA and certain proteins on human ribosome obtained using a set of mRNA analogs, oligoribonucleotide derivatives carrying alkylating or photoactivatable groups at different positions. A comparison of data on the template position on the human and Escherichia coliribosomes has revealed both the similarity in the structure of the mRNA-binding site of bacterial and mammalian ribosomes and the peculiarities of the functioning of mammalian (in particular, human) ribosomes. The similarity manifests itself in that the template codons at the A-, P-, and E-sites of bacterial and human ribosomes are surrounded by similar nucleotides (occupying similar positions in the conserved regions of secondary structure) of small subunit rRNA. The template forms a loop whose foot is in proximity to the 530 stem–loop conserved region of rRNA. The specific features of mammalian ribosomes appear to be associated with their lower conformational mobility as compared with bacterial ribosomes, owing to which their interaction with the template involves a lesser number of molecular contacts.     

mRNA periodical infrastructure complementary to the proof-reading site in the ribosome.

Virtually all mRNA sequences carry a 3-base periodical pattern, presumably involved in the translation frame monitoring mechanism (Trifonov, E.N., J. Mol. Biol. 194, 643-652, 87). The hidden pattern, 5'-(GHN)n-3' (H representing nonG, N any base), is further refined by extensive computational analysis of mRNA sequences. According to mononucleotide preferences in the three positions of coding triplets, it appears now as 5'-(GHU)n-3'. Dinucleotide frequencies independent of mononucleotides (contrast dinucleotides, 2) generate the motif 5'-(GCU)n-3'. The same motif is found by regarding the expected avoidance of destabilizing base oppositions in hypothetical transient complementary complexes between mRNA and rRNA. This hidden pattern, in its refined consensus form, 5'-(GCU)n-3', is an almost perfect complementary match to a unique site in small subunit rRNA, the universally conserved (3) proofreading loop at position 525 (of E.coli small subunit rRNA): [formula: see text] This strongly suggests that the 525 site is a major structural component of the previously proposed frame-keeping mechanism which is based on the in-frame contacts between mRNA and three segments of rRNA. Consistent with the original proposition, this site is one of three believed to interact with mRNA.     

Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens.

Protein phosphatase 2A consists of three subunits, the catalytic subunit (C) and two regulatory subunits (A and B). The A subunit has a rod-like shape and consists of 15 nonidentical repeats. It binds the catalytic subunit through repeats 11 to 15 at the C terminus and the tumor antigens encoded by small DNA tumor viruses through overlapping but distinct regions at N-terminal repeats 2 to 8. A model of the A subunit was developed on the basis of the fact that uncharged or hydrophobic amino acids are conserved at eight defined positions within each repeat. Helical wheel projections suggested that each repeat can be arranged as two interacting amphipathic helixes connected by a short loop. Mutational analysis of the A subunit revealed that the proposed loops are important for binding of tumor antigens, the B subunit, and the C subunit. Native gel analysis of mutant A subunits synthesized in vitro demonstrated that the binding region for the B subunit, previously thought to include repeats 2 to 8, covers repeats 1 to 10 and that the B and C subunits cooperate in binding to the A subunit.     

Intermolecular base-paired interaction between complementary sequences present near the 3'' ends of 5S rRNA and 18S (16S) rRNA might be involved in the reversible association of ribosomal subunits.

Highly conserved sequences present at an identical position near the 3' ends of eukaryotic and prokaryotic 5S rRNAs are complementary to the 5' strand of the m2(6)A hairpin structure near the 3' ends of 18S rRNA and 16S rRNA, respectively. The extent of base-pairing and the calculated stabilities of the hybrids that can be constructed between 5S rRNAs and the small ribosomal subunit RNAs are greater than most, if not all, RNA-RNA interactions that have been implicated in protein synthesis. The existence of complementary sequences in 5S rRNA and small ribosomal subunit RNA, along with the previous observation that there is very efficient and selective hybridization in vitro between 5S and 18S rRNA, suggests that base-pairing between 5S rRNA in the large ribosomal subunit and 18S (16S) rRNA in the small ribosomal subunit might be involved in the reversible association of ribosomal subunits. Structural and functional evidence supporting this hypothesis is discussed.