Transcription and in vitro processing of yeast 5 S rRNA

A method is described for the isolation of a yeast chromatin fraction highly enriched in ribosomal DNA sequences. In the presence of exogenous yeast RNA polymerase III, this purified chromatin actively synthesizes a set of 5 S ribosomal RNAs all of which have 5'-sequences identical with mature 5 S RNA but which end with a variable number (up to 10) of additional residues at the 3'-terminus. These extra nucleotides are precisely removed by a processing nuclease found in the chromatin supernatant fraction.     

The Kinetics of the Synthesis of Ribosomal RNA in E. coli

The kinetics of the synthesis of ribosomal RNA in E. coli has been studied using C¹⁴-uracil as tracer. Two fractions of RNA having sedimentation constants between 4 and 8S have kinetic behavior consistent with roles of precursors. The first consists of a very small proportion of the RNA found in the 100,000 g supernatant after ribosomes have been removed. It has been separated from the soluble RNA present in much larger quantities by chromatography on DEAE-cellulose columns. The size and magnitude of flow through this fraction are consistent with it being precursor to a large part of the ribosomal RNA.

A fraction of ribosomal RNA of similar size is also found in the ribosomes. This fraction is 5 to 10 per cent of the total ribosomal RNA and a much higher proportion of the RNA of the 20S and 30S ribosomes present in the cell extract. The rate of incorporation of label into this fraction and into the main fractions of ribosomal RNA of 18S and 28S suggests that the small molecules are the precursors of the large molecules. Measurements of the rate of labeling of the 20, 30, and 50S ribosomes made at corresponding times indicate that ribosome synthesis occurs by concurrent conversion of small to large molecules of RNA and small to large ribosomes.

     

Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA

In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. The primary sequence of ms RNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25 000 × g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA. Received: 17 May 1997 / Accepted: 26 August 1997     

Unusual sequences,homologous to 5S RNA,in ribosomal DNA repeats of the nematodeMeloidogyne arenaria

Summary There are sequences homologous to 5S ribosomal RNA in the ribosomal DNA (rDNA) repeats of the plant-parasitic nematodeMeloidogyne arenaria. This is surprising, because in all other higher eukaryotes studied to date, the genes for 5S RNA are unlinked to and distinct from a tandem rDNA repeat containing the genes for 18S, 5.8S, and 28S ribosomal RNA. Previously, only prokaryotes and certain lower eukaryotes (protozoa and fungi) had been found to have both the larger rRNAs and 5S rRNA represented within a single DNA repeat. This has raised questions on the organization of these repeats in the earliest cell (progenote), and on subsequent evolutionary relationships between pro- and eukaryotes.Evidence is presented for rearrangements and deletions withinMeloidogyne rDNA. The unusual life cycles (different levels of ploidy, reproduction by meiotic and mitotic parthenogenesis) of members of this genus might allow rapid fixation of any variants with introduced 5S RNA sequences. The 5S RNA sequences inMeloidogyne rDNA may not be expressed, but their presence raises important questions as to the evolutionary origins and stability of repeat gene families.     

Depth profile of sulfate-reducing bacterial ribosomal RNA and mercury methylation in an estuarine sediment

Abstract: The community structure of complex anaerobic microbial communities has been difficult to elucidate because of an inability to cultivate most of the contributing populations. In this study, the distribution of sulfate-reducing bacteria (SRB) in anaerobic sediments was determined using oligonucleotide probes complementary to the 16S ribosomal RNAs of major phylogenetic groups. Sediment cores were collected from Santa Rosa Sound in northwest Florida, and sectioned by depth into 1 to 2 cm fractions. Nucleic acids were extracted from each fraction and hybridized with the SRB-specific ribosomal RNA probes. SRB ribosomal RNAs accounted for almost 5% of the microbial community ribosomal RNA pool in the 3–4 cm depth fraction and were dominated by Desulfovibrionaceae ribosomal RNA. The SRB ribosomal RNA peak coincided with mercury methylation, an activity attributed to SRB. Profiles of the ribosomal RNAs indicate that SRB populations in sediments are stratified by depth.     

Transfer RNAs as genotypic fingerprints of eubacteria

A new method was developed for rapid genotypic identification and classification of bacteria. The method is based on high resolution gel electrophoresis of the stable, low molecular weight (LMW) RNA fraction of single bacterial strains. This fraction comprises the total transfer RNA pool and the 5S ribosomal RNA. On a one-dimensional gel, every eubacterial strain exhibited a distinct LMW RNA profile, a set of bands belonging to three different size classes: 5S rRNAs (110–131 nt), class 2 tRNAs (82–96 nt) and class 1 tRNAs (72–79 nt). LMW RNA profiles of members of five of the ten major eubacterial groups, previously defined by 16S rRNA sequence analysis, were highly diverse. For some major groups, like flavobacteria and planctomyces, the distinctive sizes of their 5S rRNAs allowed the assignment of strains to these groups. More specific taxonomic information was gained from analysis of the tRNA part of the profile. Strains could be grouped as species and genera due to species- and genus-specific tRNA bands. From an evolutionary point of view, this order found in the total tRNA pool of eubacteria could indicate that cytoplasmic tRNA evolution reflects ribosomal RNA evolution. Given the universality of tRNAs, it is to be expected that their electrophoretic mobility profiles may serve as a convenient RNA fingerprint for defining bacterial species operationally and for identifying new genotypes by differing patterns.     

Phylogeny of the 5S ribosomal RNA fromSynechococcus lividus II: The cyanobacterial/chloroplast 5S RNAs form a common structural class

Summary The complete nucleotide sequence of the 5S ribosomal RNA from the cyanobacteriumSynechococcus lividus II has been determined. The sequence is 5-UGCCUAGUGUUUAUGGCGCG-GUGGAACCACGCUGAUCCAUCCCGAACUC-AGAGGUGAAACAUCGCAGCGGUGAAGAU-AGUUGGAGGGUAGCCUCCUGCAAAAAUA-GCUCAAUGCUAGGCA_OH-3. This 5S RNA has the cyanobacterial- and chloroplast-specific nucleotide insertion between positions 30 and 31 (using the numbering system of the generalized eubacterial 5S RNA) and the chloroplast-specific nucleotide-deletion signature between positions 34 and 39. The 5S RNA ofS. lividus II has 27 base differences compared with the 5S RNA of the related strainS. lividus III. This large difference may reflect an ancient divergence between these two organisms. The electrophoretic mobilities on nondenaturing polyacrylamide gels of renatured 5S RNAs fromS. lividus II,S. lividus III, and spinach chloroplasts are identical, but differ considerably from that ofEscherichia coli 5S RNA. This most likely reflects differences in higher-order structure between the 5S RNA ofE. coli and these cyanobacterial and chloroplast 5S RNAs.     

Biosynthesis of vitamin B-12 Part I. Role of the ribosomal proteins in vitamin B-12 biosynthesis

1. 70 S ribosomes isolated from strains of Escherichia coli 113-3, K₁₂ and B take part in vitamin B-12 biosynthesis from AdoCbi-GDP, NAD and dimethylbenzimidazole in the presence of enzymes of the cytosol fraction. 2. 70 S ribosomes from E. coli 113-3 bind Ado[⁵⁸Co]Cbi-GDP. This reaction is independent of fusidic acid. 3. Proteins from 5 S RNA complex as well as L2 protein isolated from E. coli 113-3 ribosomes catalyze vitamin B-12 biosynthesis. The main catalytic function in this reaction is performed by protein L18.4. Vitamin B-12 biosynthesis proceeding in the presence of isolated ribosomal proteins is inhibited by fusidic acid, chloramphenicol and vernamycin but not by erythromycin. 5. Vitamin B-12 synthesized in the presence of isolated ribosomal proteins is biologically active.     

Evolution of green plants and their relationship with other photosynthetic eukaryotes as deduced from 5S ribosomal RNA sequences

The nucleotide sequence of cytoplasmic 5S ribosomal RNAs from three gymnosperms,Pinus contorta, Taxus baccata andJuniperus media and from one fern,Pteridium aquilinum, have been determined. These sequences were aligned with all hitherto known cytoplasmic 5S ribosomal RNA sequences of photosynthetic eukaryotes. A dendrogram based on that set of sequences was constructed by a distance matrix method and the resulting tree compared with established views concerning plant and algal evolution. The following monophyletic groups of photosynthetic eukaryotes are recognizable: theRhodophyta, a group consisting ofPhaeophyta, Bacillariophyta andChrysophyta, and the green plants, the latter comprising green algae,Bryophyta, Pteridophyta andSpermatophyta. According to our 5S ribosomal RNA tree, green plants may have originated from some type of a green flagellated organism such asChlamydomonas. The land plants seem to have originated from some form of charophyte such asNitella. 5S ribosomal RNA seems to be less appropriate to estimate dissimilarities between species which have diverged relatively recently, like the angiosperms. Therefore, a precise evolutionary process is difficult to reconstruct for members of this group.     

Phylogenetic origins of the plant mitochondrion based on a comparative analysis of 5S ribosomal RNA sequences

Summary The complete nucleotide sequences of 5S ribosomal RNAs fromRhodocyclus gelatinosa, Rhodobacter sphaeroides, andPseudomonas cepacia were determined. Comparisons of these 5S RNA sequences show that rather than being phylogenetically related to one another, the two photosynthetic bacterial 5S RNAs share more sequence and signature homology with the RNAs of two nonphotosynthetic strains.Rhodobacter sphaeroides is specifically related toParacoccus denitrificans andRc. gelatinosa is related toPs. cepacia.These results support earlier 16S ribosomal RNA studies and add two important groups to the 5S RNA data base. Unique 5S RNA structural features previously found inP. denitrificans are present also in the 5S RNA ofRb. sphaeroides; these provide the basis for subdivisional signatures. The immediate consequence of our obtaining these new sequences is that we are able to clarify the phylogenetic origins of the plant mitochondrion. In particular, we find a close phylogenetic relationship between the plant mitochondria and members of the alpha subdivision of the purple photosynthetic bacteria, namely,Rb. sphaeroides, P. denitrificans, andRhodospirillum rubrum.     

Ribonucleic Acid-Dependent Ribonucleic Acid Polymerase in the Immune Response

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 M column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA preparation (iRNA) as template made from the spleens of immunized mice but very low activity was found with an nRNA preparation made from the spleens of normal mice. Incorporation of ³H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12–13 S was most active as a template. It was followed by a fraction corresponding to 6–7 S. Sucrose gradient analysis of the ³H-UTP-labeled product was attempted. Some properties of this enzyme are described.     

Fragments of rDNA within the Chinese hamster genome

We report the isolation and partial characterization of distinctEcoRI fragments of the Chinese hamster genome which contain regions complementary to a 1-kb portion of the mature 18 S ribosomal RNA molecule. This previously undescribed 18 S rDNA-like region, which we have termed a fragment of ribosomal DNA (frDNA), has been shown by sequence analysis to correspond to a region extending 1 kb upstream from the 3 terminus of the mature 18 S rRNA. Within the five frDNA-containing clones described here, no other region of the ribosomal RNA cistron was detected, making it unlikely that these are polymorphic forms of the ribosomal DNA repeat. The 18 S rDNA-complementary region appears to be flanked by an imperfect direct repeat, which could have been the result of the retroinsertion of a fragment of ribosomal RNA. Directly adjacent to the 18 S rDNA-like region we have identified nonribosomal sequences which appear common to all of the frDNA-containing clones we examined. At least eight different-sizedEcoRI fragments contain frDNAs and the abundance of the frDNAs appears to be of the order of 30 per genome. The occurrence of multiple copies of this ribosomal-nonribosomal chimera suggests that, once formed, the chimera was duplicated within the genome.This work was supported by funds provided by the Graduate School of the University of Wisconsin—Milwaukee, the Cancer Center of the Medical College of Wisconsin, and the Shaw Scholars program of the James D. Shaw and Dorothy Shaw Fund, Milwaukee Foundation.     

Poly(A)+ RNA from Tetrahymena: Stimulation of Protein Synthesis in Vitro*†

SYNOPSIS Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at -17S and 25S. After heat denaturation all the 25S RNA was converted to 17S. indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A)–RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15–fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28.000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

On the biosynthesis of bacterial ribosomal RNA

By comparison of the fingerprints of 5S and 23S ribosomal RNAs from Bacillus licheniformis with that of the precursor of 23S ribosomal RNA, it can be shown that 5S RNA is not a part of the precursor of 23S ribosomal RNA.     

Studies of newly synthesized ribosomal ribonucleic acid of Escherichia coli

1. RNA synthesized by Escherichia coli during one-hundredth of the generation time contains two fractions distinguishable by hybridization with homologous DNA. One fraction, approximately 30% of the newly synthesized RNA, did not compete with ribosomal RNA, being apparently messenger RNA. The other fraction, approximately 70% of the newly made RNA, hybridized as ribosomal RNA. These values are comparable with previous estimates (McCarthy & Bolton, 1964; Pigott & Midgley, 1968). 2. Hybridization-competition experiments showed that the newly made RNA associated with 70s ribosomes and larger ribosome aggregates was a mixture of ribosomal RNA and messenger RNA, whereas that associated with nascent ribosomal subunits consisted exclusively of ribosomal RNA. This observation provides means by which newly synthesized ribosomal RNA can be isolated free from messenger RNA. 3. Newly made ribosomal RNA in nascent ribosomal subunits was sensitive to shear under conditions where ribosomal RNA in mature ribosomes was shear-resistant. Thus, when RNA was extracted from cells of E. coli disrupted by mechanical means, newly made ribosomal RNA appeared heterogeneous in size, sedimenting as a broad peak extending from 8s to 16s. 4. Newly synthesized ribosomal RNA in nascent ribosomal subunits was rapidly degraded in the presence of actinomycin D and during glucose starvation. 5. Newly synthesized ribosomal RNA stimulated amino acid incorporation in a system synthesizing protein in vitro to the same extent as the RNA which contained the messenger RNA fraction.     

RNase-sensitive DNA polymerase activity in cell fractions and mutants of Neurospora crassa

RNase-sensitive DNA polymerase activity (RSDP) was tested in different cell fractions of Neurospora crassa cell types and its morphological mutants. This RSDP was found localized in the microsomal pellet fraction and absent in the purified nuclear pellets isolated from different N. crassa cell types: conidia, germinated conidia, and mycelia. This enzyme is capable of synthesizing a DNA product only in the presence of all four deoxyribonucleoside-5-triphosphates and Mg²⁺. Removal of RNA from the pellet fraction by RNase strongly inhibited the DNA synthesis. The endogenous synthesis of DNA in the microsomal pellet fraction was associated with the formation of an RNA:DNA hybrid as analyzed by Cs₂SO₄ equilibrium density gradient centrifugation. The DNA product after alkali hydrolysis hybridizes with the RNA isolated from the same pellet fraction, as analyzed by elution from hydroxylapatite column at 60 C. This DNA product did not hybridize with poly(A). A few mutants tested showed this RNase-sensitive DNA polymerase activity.This work was supported in part by a contract with the U.S. Department of Energy and a grant from the U.S. Naval Research.     

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5′ untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism.