Characterization of a separate small domain derived from the 5' end of 23S rRNA of an alpha-proteobacterium.

We demonstrate the presence of a separate processed domain derived from the 5' end of 23S rRNA in ribosomes of Rhodopseudomonas palustris, a member of the alpha-++proteobacteria. Previous sequencing studies predicted intervening sequences (IVS) at homologous positions within the 23S rRNA genes of several alpha-proteobacteria, including R.palustris, and we find a processed 23S rRNA 5' domain in unfractionated RNA from several species. 5.8S rRNA from eukaryotic cytoplasmic large subunit ribosomes and the bacterial processed 23S rRNA 5' domain share homology, possess similar structures and are both derived by processing of large precursors. However, the internal transcribed spacer regions or IVSs separating them from the main large subunit rRNAs are evolutionarily unrelated. Consistent with the difference in sequence, we find that the site and mechanism of IVS processing also differs. Rhodopseudomonas palustris IVS-containing RNA precursors are cleaved in vitro by Escherichia coli RNase III or a similar activity present in R.palustris extracts at a processing site distinct from that found in eukaryotic systems and this results in only partial processing of the IVS. Surprisingly, in a reaction unlike characterized cases of eubacterial IVS processing, an RNA segment larger than the corresponding DNA insertion is removed which contains conserved sequences. These sequences, by analogy, serve to link the 23S rRNA 5' rRNA domains or 5.8S rRNAs to the main portion of other prokaryotic 23S rRNAs or to eukaryotic 28S rRNAs, respectively.     

Phylogenetic position of someChlorella species within the chlorococcales based upon complete small-subunit ribosomal RNA sequences

Summary Complete small-subunit rRNA (16S-like rRNA) coding region sequences were determined for eight species of the Chlorococcales (Chlorophyceae). The genera investigated includePrototheca, Ankistrodesmus, Scenedesmus, and fiveChlorella species. Distance matrix methods were used to infer a phylogenetic tree that describes evolutionary relationships between several plant and green algal groups. The tree exhibits a bifurcation within the Chlorococcales consistent with the division into Oocystaceae and Scenedesmaceae, but three of the fiveChlorella species are more similar to other algae than toChlorella vulgaris. All of the sequences contain primary and secondary structural features that are characteristic of 16S-like rRNAs of chlorophytes and higher plants.Anikstrodesmus stipitatus, however, contains a 394-bp group I intervening sequence in its 16S-like rRNA coding region.     

Nuclease S1 mapping of 16S ribosomal RNA in ribosomes

Escherichia coli 16S rRNA and 16S-like rRNAs from other species have several universally conserved sequences which are believed to be single-stranded in ribosomes. The quantitative disposition of these sequences within ribosomes is not known. Here we describe experiments designed to explore the availability of universal 16S rRNA sequences for hybridization with DNA probes in 30S particles and 70S ribosomes. Unlike previous investigations, quantitative data on the accessibility of DNA probes to the conserved portions of 16S rRNA within ribosomes was acquired. Uniquely, the experimental design also permitted investigation of cooperative interactions involving portions of conserved 16S rRNA. The basic strategy employed ribosomes, 30S subunits, and 16S rRNAs, which were quantitatively analyzed for hybridization efficiency with synthetic DNA in combination with nuclease S1. In deproteinated E. coli 16S rRNA and 30S subunits, the regions 520-530, 1396-1404, 1493-1504, and 1533-1542 are all single-stranded and unrestricted for hybridization to short synthetic DNAs. However, the quantitative disposition of the sequences in 70S ribosomes varies with each position. In 30S subunits there appear to be no cooperative interactions between the 16S rRNA universal sequences investigated.     

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.     

Estimating Substitution Rates in Ribosomal RNA Genes

A model is introduced describing nucleotide substitution in ribosomal RNA (rRNA) genes. In this model, substitution in the stem and loop regions of rRNA is modeled with 16- and four-state continuous time Markov chains, respectively. The mean substitution rates at nucleotide sites are assumed to follow gamma distributions that are different for the two types of regions. The simplest formulation of the model allows for explicit expressions for transition probabilities of the Markov processes to be found. These expressions were used to analyze several 16S-like rRNA genes from higher eukaryotes with the maximum likelihood method. Although the observed proportion of invariable sites was only slightly higher in the stem regions, the estimated average substitution rates in the stem regions were almost two times as high as in the loop regions. Therefore, the degree of site heterogeneity of substitution rates in the stem regions seems to be higher than in the loop regions of animal 16S-like rRNAs due to presence of a few rapidly evolving sites. The model appears to be helpful in understanding the regularities of nucleotide substitution in rRNAs and probably minimizing errors in recovering phylogeny for distantly related taxa from these genes.     

An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence.

Regions extremely variable in size and sequence occur at conserved locations in eukaryotic rRNAs. The functional importance of one such region was determined by gene reconstruction and replacement in Tetrahymena thermophila. Deletion of the D8 region of the large-subunit rRNA inactivates T. thermophila rRNA genes (rDNA): transformants containing only this type of rDNA are unable to grow. Replacement with an unrelated sequence of similar size or a variable region from a different position in the rRNA also inactivated the rDNA. Mutant rRNAs resulting from such constructs were present only in precursor forms, suggesting that these rRNAs are deficient in either processing or stabilization of the mature form. Replacement with D8 regions from three other organisms restored function, even though the sequences are very different. Thus, these D8 regions share an essential functional feature that is not reflected in their primary sequences. Similar tertiary structures may be the quality these sequences share that allows them to function interchangeably.     

Sequence analysis of the transcribed and 5'' non-transcribed regions of the ribosomal RNA gene in Dictyostelium discoideum

The nucleotide sequence of Dictyostelium discoideum rDNA extending over almost the entire transcribed region and a part of the 5' non-transcribed spacer region has been determined. Computer analysis revealed that there were several conserved sequences in the 17S, 5.8S and 26S coding regions when compared with the sequences at analogous positions in some eukaryotic rRNA genes. The data also showed that the D. discoideum rDNA contains several extra sequences, which have not been found in other eukaryotes' rDNAs , near the 3' terminus of the 17S coding region and the 5' terminus of the 26S coding region.     

Homology of the 3'' terminal sequences of the 18S rRNA of Bombyx mori and the 16S rRNA of Escherchia coli.

The terminal 220 base pairs (bp) of the gene for 18S rRNA and 18 bp of the adjoining spacer rDNA of the silkworm Bombyx mori have been sequenced. Comparison with the sequence of the 16S rRNA gene of Escherichia coli has shown that a region including 45 bp of the B. mori sequence at the 3' end is remarkably homologous with the 3' terminal E. coli sequence. Other homologies occur in the terminal regions of the 18S and 16S rRNAs, including a perfectly conserved stretch of 13 bp within a longer homology located 150--200 bp from the 3' termini. These homologies are the most extensive so far reported between prokaryotic and eukaryotic genomic DNA.     

18S coding sequences in amplified ribosomal DNA from Xenopus laevis oocytes are highly homogeneous, unmethylated, and lack major open reading frames.

We have used two approaches to search for sequence variants in the 18S coding region of amplified ribosomal DNA (rDNA) from Xenopus laevis oocytes. First, using clones derived from amplified rDNA, we compared the equivalent of a complete 18S coding region from two clones and short regions from two other clones with the 18S sequence previously determined from a "reference" clone. The respective sequences in all the clones were identical. Secondly, we examined greater than 60% of the 18S sequence in "pooled 18S genes" in uncloned amplified rDNA. The predominant sequence corresponded to that in the reference clone and no heterogeneities were apparent. Since many chromosomal rDNA units contribute to rDNA amplification the findings indicate that 18S coding sequences in X. laevis are largely homogeneous. The previously established sequence is the predominant one, thus providing a reliable basis for studies on 18S rRNA. Sequencing gels on uncloned amplified rDNA confirmed the absence of methylated cytosine in this DNA. The 18S sequence lacks major open reading frames.     

Sequence and secondary structure of Drosophila melanogaster 5.8S and 2S rRNAs and of the processing site between them.

Drosophila melanogaster 5.8S and 2S rRNAs were end-labeled with 32p at either the 5' or 3' end and were sequenced. 5.8S rRNA is 123 nucleotides long and homologous to the 5' part of sequenced 5.8S molecules from other species. 2S rRNA is 30 nucleotides long and homologous to the 3' part of other 5.8S molecules. The 3' end of the 5.8S molecule is able to base-pair with the 5' end of the 2S rRNA to generate a helical region equivalent in position to the "GC-rich hairpin" found in all previously sequenced 5.8S molecules. Probing the structure of the labeled Drosophila 5.8S molecule with S1 nuclease in solution verifies its similarity to other 5.8S rRNAs. The 2S rRNA is shown to form a stable complex with both 5.8S and 26S rRNAs separately and together. 5.8S rRNA can also form either binary or ternary complexes with 2S and 26S rRNA. It is concluded that the 5.8S rRNA in Drosophila melanogaster is very similar both in sequence and structure to other 5.8 rRNAs but is split into two pieces, the 2S rRNA being the 3' part. 2S anchors the 5.8S and 26S rRNA. The order of the rRNA coding regions in the ribosomal DNA repeating unit is shown to be 18S - 5.8S - 2S - 26S. Direct sequencing of ribosomal DNA shows that the 5.8S and 2S regions are separated by a 28 nucleotide spacer which is A-T rich and is presumably removed by a specific processing event. A secondary structure model is proposed for the 26S-5.8S ternary complex and for the presumptive precursor molecule.     

Structure and sequence of the mitochondrial 20S rRNA and tRNA tyr gene of Paramecium primaurelia

The sequence and structure of the large (20s) mitochondrial (mt) rRNA gene and flanking regions from Paramecium primaurelia have been determined. The gene contains two regions of strong homology with other large mt rRNAs: one 44-base region near the 5' end and a 321-base region near the 3' end. Another region of strong homology to both ends of E. coli 23s RNA exists at loci consistent with these regions. The Paramecium gene appears to be 2204 bases in length and contains slightly more homology to E. coli rRNA than its mammalian or fungal counterparts. The gene, located about 1200 bp from the replicative terminal end of the linear mt DNA, is transcribed in the same polarity as replication. Previous R-looping studies detected no large introns within the gene. Here we describe sequences resembling degenerate rRNAs, one of which could represent a small intron. A tRNA tyr gene was found on the same DNA strand, 127 bp downstream from the large rRNA presumptive 3' end. The tRNA is flanked on both sides by short DNA regions of approximately 90% A + T content.     

The ribosomal RNA genes from chloroplasts of mustard (Sinapis alba L.): mapping and sequencing of the leader region

Summary The genes coding for rRNAs from mustard chloroplasts were mapped within the inverted repeat regions of intact ctDNA and on ctDNA fragments cloned in pBR322. R-loop analysis and restriction endonuclease mapping show that the genes for 16S rRNA map at distances of 17 kb from the junctions of the repeat regions with the large unique region. The genes for 23S rRNA are located at distances of 2.8 kb from the junctions with the small unique region. Genes for 4.5S and 5S rRNA are located in close proximity to the 23S rRNA genes towards the small unique region. DNA sequencing of portions of the 5 terminal third from the mustard 16S rRNA gene shows 96–99% homology with the corresponding regions of the maize, tobacco and spinach chloroplast genes. Sequencing of the region proximal to the 16S rRNA gene reveals the presence of a tRNA^Val gene in nearly the same position and with identical sequence as in maize, tobacco and spinach. Somewhat less but still strong homology is also observed for the tDNA ^Val/16S rDNA intercistronic regions and for the regions upstream of the tRNA^Val gene. However, due to many small and also a few larger deletions and insertions in the leader region, common reading frames coding for homologous peptides larger than 44 amino acids can not be detected; it is therefore unlikely that this region contains a protein coding gene.