Ribosomal RNA Components Research Articles

Cytoplasmic zone analysis. RNA flow studied by micromanipulation.

A technique is described, cytoplasmic zone analysis, by which it is possible to study the flow of different RNA-containing components in the cytoplasm after their release from the nucleus. After a pulse of RNA precursors, the salivary glands of the insect Chironomus tentans are fixed and microdissected for the isolation of three zones of cytoplasm situated at increasing distances from the nucleus. The RNA from each zone is isolated and analyzed by gel electrophoresis. The three ribosomal RNA components, 18 S, 28 S and 5 S RNA, appear in steep, specific radioactivity gradients (exit gradients) during the time interval 2-3 h after a precursor injection, the 18 S RNA gradient lying 30-50 mum peripheral to that of the 28 S or 5 S RNA gradient. Administration of puromycin led to the complete disappearance of the 28 S RNA and most of the 28 S RNA gradient within 45 min, suggesting that the gradients are caused by an engagement of the ribosomal subunits into polysomes close to the nucleus immediately or soon after their exit from the nucleus. The gradients may offer a unique model for the study of polysome formation and maintenance in vivo.

Transcription and Processing of Transfer RNA Precursors

This chapter discusses the transcription of tRNA genes as precursors and how these are converted to mature tRNA. One important step in tRNA biosynthesis, the modification of tRNA bases, is discussed only in relation to the precursors. In the chapter, the term precursor is used to indicate the original transcript or any intermediate in the size reduction of this to tRNA, processing denotes any of the nucleolytic cleavages in these steps, and the term modification is reserved for the various substitutions and rearrangements that yield the modified nucleoside residues in tRNA. The primary transcripts are longer RNAs containing extra sequences not present in the functional molecules. In the case of the mammalian ribosomal RNA precursor, three separate ribosomal RNA components are cotranscribed. The chapter reviews that the tRNA precursors were first found in mammalian cells, but most of the knowledge about these steps in the biosynthesis of tRNA has come from Escherichia coli, where a combination of genetic analysis and nucleotide sequencing not only has revealed much about how the tRNAs are transcribed, but also has led to the discovery of a set of highly specific nucleases involved in the cleavage of the tRNA gene transcripts.

Migration of ribosomes along the axons of the chick visual pathway

The axoplasmic migration of ribosomes has been detected in the visual system of the chick. Monocular injection of radioactive uridine or an amino acid mixture was followed by sedimentation analysis in sucrose or cesium sulfate density gradients, of ribosomes prepared from the retinae of injected eyes and the left and right optic lobes. By this means both RNA and protein components of ribosomes were found to migrate from the retina to the innervated contralateral optic lobe. Following denervation of the distal nerve segment by eye removal, the stability of the transported RNA was reduced, suggesting its presynaptic location. The transport of RNA was not significantly impaired by intraocular injection of inhibitors of informational RNA or mitochondrial RNA synthesis prior to injection of radioactive uridine but was depressed by a low dose of actinomycin D.

In vivo labeling of cytoplasmic RNA in neurons of the immature brain cortex

only the total and the nuclear RNAs were analyzed. The present report describes a procedure for the isolation of the neuronal cytoplasmic RNA and its application to the measurement of the approximate maturation time of its ribosomal RNA component in 8- and 20-day-old cerebral cortex perikarya. While this work was in progress a report describing the labeling pattern of neuronal and glial cytoplasmic RNA isolated from the whole brain of adult rats has appeared ~. Neuronal perikarya were isolated as described previously, except for the following modification: the solution employed for tissue suspension contained 7.5 ~ polyvinylpyrrolidone (w/v), 10 mM CaC12 and 4 ~ (w/v) instead of the 1 ~ bovine serum albumin previously used 1°. With this change in albumin concentration the suspension solution becomes isotonic. The yield and preservation of the perikaryal fraction and its purity were described 9,1°. In each experiment 10 rats were injected intrathecally with 10/~Ci of [5-aH]uridine (Amersham/Searle, 20 Ci/mmole). The neuronal perikarya were suspended in 3 ml of a solution consisting of 0.2 M sucrose- 0.06 M NaCI0.1 M Tris-HC1 buffer, pH 7.6, and the suspension was homogenized by means of 3 strokes in a glass-teflon loose-fitting homogenizer. The homogenate was centrifuged at 2,000 × g for 10 min and the supernatant was aspirated and stored in ice. This procedure was repeated and the supernatant pool was brought up to 0.25 ~ Macaloid (American Tansul Co., Houston, Texas), 0.01 M EDTA and 50/~g/ml of polyvinyl

Stimulation of ribosomal and DNA-like RNA synthesis by α-methyltryptophan

Rapidly labeled nuclear RNA from rat liver was fractionated by methyl albumin kieselguhr chromatography into the ribosomal and DNA-like RNA components. Injection of 100 mg/kg of α-methyltryptophan into rats stimulated the labeling by [ 3H] orotic acid of both species of nuclear RNA; there was a greater effect on the ribosomal than on the RNA-like fraction. This stimulatory effect of α-methyltryptophan could not be accounted for by an increase in the uptake of the labeled nucleoside by the liver, nor by the increase in the specific activity of the nucleoside and nucleotide pool of the liver.

‘2 S’ RNA, A new ribosomal RNA component in cultured Drosophila cells

FEBS LettersVolume 44, Issue 1 p. 39-42 Full-length articleFree Access ‘2 S’ RNA, A new ribosomal RNA component in cultured Drosophila cells B.R. Jordan, B.R. Jordan CNRS—Centre de Biochimie et de Biologie Moléculaire 31, chemin Joseph Aiguier 13274 Marseille, Cédex 2, FranceSearch for more papers by this author B.R. Jordan, B.R. Jordan CNRS—Centre de Biochimie et de Biologie Moléculaire 31, chemin Joseph Aiguier 13274 Marseille, Cédex 2, FranceSearch for more papers by this author First published: August 15, 1974 https://doi.org/10.1016/0014-5793(74)80301-7Citations: 15AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 J. Pene, E. Knight, J.E. Darnell, J. Mol. Biol., 33, (1968), 609– 623. 2 S.A. Udem, K. Kaufman, R.J. Warner, J. Bacteriol., 105, (1971), 101– 106. 3 S.A. Udem, J.R. Warner, J. Mol. Biol., 65, (1972), 227– 242. 4 G. Echalier, A. Ohanessian, Vitro, 6, (1970), 162– 172. 5 U. Loening, Biochem. J., 113, (1969), 131– 138. 6 A.E. Dahlberg, C.W. Dingman, A.C. Peacock, J. Mol. Biol., 41, (1969), 139– 147. 7 B.R. Jordan, J. Mol. Biol., 55, (1971), 423– 439. 8 G.G. Brownlee, F. Sanger, B.G. Barrell, J. Mol. Biol., 34, (1968), 379– 412. 9 J. Shine, L. Dalgarno, J. Mol. Biol., 75, (1973), 57– 72. Citing Literature Volume44, Issue1August 15, 1974Pages 39-42 ReferencesRelatedInformation

Minor components of rat liver ribosomal RNA as possible intermediates in the degradation of 28S RNA in vivo effects of partial hepatectomy and starvation on their relative amounts.

The minor RNA components in large ribosomal subunits of rat liver were analyzed by gel electrophoresis. Quantitative analysis showed that these minor components, whose apparent molecular weights ranged from 8.48 × 105 to 11.4 × 105, represented 10 to 13% of the total high-molecular-weight ribosomal RNA.It was elucidated that they were not artifacts arose during the cell homogenization, sub-cellular fractionation, RNA preparation and gel electrophoresis.Labeling experiment in vivo showed that they were preferentially present in “old” ribosomes. It was predicted that these minor components were the intermediates in the degradation of 28S ribosomal RNA in vivo.In the regenerating liver, where the degradation of proteins was reported to be blocked, the relative amount of the minor RNA components was decreased to about a half of that of normal liver. There was, however, no increase in their relative amount in the long-term starved rat liver, where RNA degradation should be going very rapidly.

Ribosomal RNA cistrons in Euglena gracilis

Euglena gracilis chloroplasts contain about 12 fg DNA of average density 1.686 g cm −3 and 1.7 pg RNA. The large (1.1 × 10 6 mol. wt) and small (0.56 × 10 6 mol. wt) ribosomal RNA components are coded for by separate cistrons, both of which band at a density of 1.696 g cm −3 in a CsCl gradient. About 6% of the chloroplast DNA codes for rRNA indicating that there are 240 cistrons for rRNA in each chloroplast or about three to six cistrons per chloroplast genome. Similar studies with rRNA from cytoplasmic ribosomes indicate that the cistrons for cytoplasmic rRNA band at a density of 1.716 g cm −3, denser than that of the main-band DNA, and that there are 1000 cistrons for cytoplasmic rRNA per cell. Fractionation of E. gracilis DNA on CsCl gradients and subsequent hybridization experiments, as well as melting curves of DNA-RNA hybrids, show that chloroplast rRNA does not anneal specifically with either the cistrons for cytoplasmic rRNA or any DNA in the dark-grown cell, in contrast to those results found in some higher plants.

Assembly of Escherichia coli 50 S Ribosomes from Ribonucleic Acid and Protein Components: I. CHEMICAL AND PHYSICAL FACTORS AFFECTING THE CONFORMATION OF ASSEMBLED PARTICLES

Abstract The in vitro assembly of Escherichia coli 50 S ribosomes from the ribonucleic acid and protein components of 50 S ribosomes was attempted. The two components were prepared by subjecting 50 S ribosomes to LiCl-urea treatment. The ribonucleic acid fraction contains 23 S RNA and 5 S RNA in equimolar amounts and is free from significant amounts of protein. The protein fraction is composed of all of the protein subunits of 50 S ribosomes. The reconstitution experiments were carried out in a standard system: 0.01 m Tris HCl, pH 7.5–0.3 m KCl-0.02 m magnesium acetate-0.006 m β-mercaptoethanol. The ribosomal RNA and protein components were incubated in the standard system at 42° for 15 min. These conditions permitted the formation of 30 S ribosomes from their components with 72% recovery of the original activity, as measured by polyuridylic acid-directed phenylalanine incorporation. Under identical conditions, the RNA and protein components of 50 S ribosomes were assembled stoichiometrically into 37 S particles. In a search for a chemical agent that would effectively reassemble particles of more compact structure, we found that spermine or spermidine or both, in addition to Mg++, led to the formation of 45 to 48 S ribonucleoprotein particles with all of the macromolecular components of 50 S ribosomes. The 45 to 48 S particles and 37 S particles were inactive in terms of polyuridylic acid-directed phenylalanine incorporation, the peptidyltransferase reaction and peptide chain termination reaction. We also showed that 23 S ribonucleoprotein particles are assembled in the absence of Mg++ and polyamines. The particles continuously increase in sedimentation constant from 23 S to 26 S when Mg++ is gradually increased to 10-2 m. Above 10-2 m Mg++, 36 S particles appear and the sedimentation constant of the particles plateaus at 37 to 39 S at the concentration higher than 2 x 10-2 m Mg++. Spermine or spermidine, in addition to Mg++, further increased the sedimentation constant of the particles continuously to 45 to 48 S. The process is the reversal of the previously reported unfolding of 50 S ribosomes by citrate-Mg++ treatment. NH4Cl, in place of KCl, or the pH in a range between 7 and 8 had no significant effects on the ribosomal assembly. Mg++ or Mg++ plus polyamine(s) have to be added to the assembly system at zero time. The later those cations are added, the more unfolded are the resultant particles. The incorporation of 5 S RNA into the assembled ribonucleoprotein particles is stoichiometric and highly specific so that no significant incorporation of tRNA was observed.

Differentiation of 23-S ribosomal RNA cistrons from 16-S ribosomal RNA cistrons in Escherichia coli by the sequence complementarity test

It has been reported that the 16-S and 23-S components of Escherichia coli ribosomal RNA compete for common hybridization sites on homologous DNA. We here show that no such competition phenomenon occurs. Carefully purified preparations of the two major ribosomal RNA types do not competitively interact with one another during hybridization to Escherichia coli DNA. Consequently, as in all other organisms tested to date, genes coding for molecules of 23-S ribosomal RNA are entirely distinct from those giving rise to 16-S ribosomal RNA molecules. We also confirm that there are multiple gene copies responsible for the biogenesis of each ribosomal RNA component in Escherichia coli.

Characterization of ribosomes from the seed of Pinus lambertiana

Ribosomes isolated from seeds of the sugar pine, Pinus lambertiana, have been characterized: The ribosome has a sedimentation coefficient ( s 20,w 0) of 78·2 S and contains 41 % RNA and 58 % protein. On dialysis against buffer containing 0·5-1 mM MgCl 2, the ribosome was reversibly transformed into an intermediate form (60 S). Further removal of Mg 2+ causes the intermediate ribosome to dissociate into subunits (30 S and 40 S). Treatment of the intermediate ribosome with p-chloromercuribenzoic acid caused the dissociation of the particle into subunits. Incubating the 80 S ribosome with the sulfhydryl reagent caused a rapid transformation of the particle into an intermediate type particle. These results suggest that sulfhydryl groups are involved not only in associating the subunits but also in maintaining the compact structure of the ribosomes. The ribosome contains three ribosomal RNA components of 28 S, 18 S and 5 S. The base compositions of the three ribosomal RNA components are different.

Structural comparison of 37-S and 32-S ribosomal precursor RNA in yeast with the mature ribosomal RNA components

Fingerprints were made of Me- 3H-labeled 32-S and 37-S ribosomal precursor RNA plus the two Me- 14C-labeled rRNA components of yeast. The results show that both 32-S and 37-S RNA contain methylated oligonucleotide sequences in common with the two rRNAs. This confirms the assumption expressed earlier (Retèl, J. and Planta, R. J. (1970) Biochim. Biophys. Acta 224, 458) that 32-S RNA and 37-S RNA are common precursors of both rRNAs.

Terminal nucleotides of avian myeloblastosis virus RNA and of ribosomal RNA from chicken leukemic myeloblasts

The 18- and 28-S ribosomal RNA components from chicken leukemic myeloblasts have been purified and separately analyzed for terminal nucleotides. Alkaline hydrolysates of 18-S rRNA contain, per mole RNA, about 1 mole of cytidine 3′(2′),5′-diphosphate and 1 mole of adenosine. Similar analysis of the 28-S component gives only guanosine 3′(2′),5′-diphosphate and uridine. Fractionation of nuclease hydrolysates of each RNA by a “diagonal” electrophoretic method (which exploits the absence of monoesterified phosphate on the 3′-terminal oligonucleotide) permits isolation of a terminal fragment from each. Analysis of these fragments indicates that the terminal nucleotide sequence of myeloblast 18-S rRNA is -GC[(AU) 2C 2U 2]A OH and that of the 28-S rRNA is -PyAGGU OH. Analysis of the nucleosides released upon alkaline hydrolysis of avian myeloblastosis virus RNA show the 3′-terminus to be predominantly adenosine.

The pattern of methylation of RNA in peripheral nerve of the chick during development.

AbstractThe pattern of the methylation of RNA was investigated in organ cultures of the sciatic nerve of the chicken. Nerve tissue from 14‐day embryos, 17‐day embryos and 3‐day‐ old chicks was incubated with [methyl‐3H]methionine or with [2‐14C]uridine and [methyl‐3H]methionine simultaneously for various periods of time. Subsequently, RNA was extracted from the tissues and the purified preparations were fractionated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the rapidly labelled RNA changed during the three developmental stages. The incorporation of both uridine and the methyl groups from methionine was highest in the‘heavy’RNA species of the 14‐day embryonic nerve during the 0.5 and 1.0 h incubation periods. In contrast, in the nerves of 3‐day‐old chicks during a 0.5 h pulse with both precursors, methylation was almost entirely limited to the transfer RNA species. Furthermore, the incorporation of uridine in the nerves from 3‐day‐old animals revealed the presence of a heterogeneous population of rapidlylabelled, unmethylated species of RNA, most of which migrated between the smaller ribosomal RNA and transfer RNA components of the bulk RNA. The pattern of uridine incorporation and the methylation of the rapidly‐labelled RNA of the 17‐day embryonic nerve represented a transitional state between that of the 14‐day embryos and that of the 3‐day‐old chicks. The 17‐day embryonic stage of development corresponded to the phase of the onset of rapid deposition of myelin lipids in the sciatic nerve. Pulse‐chase experiments on the embryonic nerves indicated that a number of methylated precursors of ribosomal RNA and labile, heterogeneous, probably DNA‐like RNA were synthesized.

The production of ribosomal RNA from high molecular weight precursors: III. Hydrolysis of pre-ribosomal and ribosomal RNA by a 3′-OH specific exoribonuclease

Highly purified preparations of a nuclear exoribonuclease were used to hydrolyze individual ribosomal and pre-ribosomal RNA components. The kinetics of hydrolysis of 45, 32, 28 and 18 s ribosomal RNA were studied and the frequency of methylated nucleotides near the 3′-OH termini of the various rRNA components was determined. The results indicate that there are methyl-deficient segments greater than 600 nucleotides long at the 3′-OH ends of both the 45 s and 32 s molecules, and that these segments can be hydrolyzed by the exonuclease at rates which are at least an order of magnitude greater than those observed for the mature 28 s and 18 s species. There is a great deceleration in rate, however, when the enzyme reaches certain internal points within the precursor molecules. The foregoing data are discussed in regard to the possible arrangements of the 28 s and 18 s segments within the 45 s molecule and to the possible enzymic mechanisms for rRNA processing.

Characterization of the ribosomal ribonucleic acids of blue-green algae.

The ribosomal RNA components of 12 species of blue-green algae have been characterized. The 23S RNA of most species is labile and discrete cleavage products were detected by polyacrylamide gel electrophoresis. In contrast, the 23S and 16S RNA's of three species, Anacystis nidulans, Nostoc sp. and Oscillatoria tenuis were essentially undegraded (apart from a “hidden” break in some of the 23S RNA molecules) and these are the most suitable species for further study. The undegraded 23S and 16S RNA's have similar molecular weights (1.07×106 and 0.53–0.54×106 respectively) to the corresponding molecules from bacteria and eukaryote chloroplasts. The nucleotide base compositions of separated, intact, 23S and 16S RNA's from blue-green algae are also of the prokaryotic type. For instance, the (G+C) content of each RNA is approximately 52 moles % and the (G-C)+(A-U) values are high (16–24 moles %). Blue-green algae, like other organisms, contain a 5S ribosomal RNA. Its electrophoretic mobility in polyacrylamide gels and its behaviour on methylated-albumen-kieselguhr-columns relative to E. coli, plant cytoplasmic and plant chloroplast 5S RNA's, are described.

Expression of the mitochondrial genome in Hela cells: VI. Size determination of mitochondrial ribosomal RNA by electron microscopy

A modified basic protein, film method of spreading RNA in a strongly denaturing solvent for examination in the electron microscope has been developed and applied to determine the size of the HeLa mitochondrial specific ribosomal RNA components. Length measurements on purified 12 s and 16 s mitochondrial rRNA, on mixtures of the two, and on mixtures of 12 s with 18 s cytoplasmic rRNA, have given molecular lengths of 0.27 μ, 0.42 μ, and 0.55 μ for the 12 s, 16 s and 18 s rRNA's. If these molecular lengths are proportional to molecular weight, and if the molecular weight of 18 s cytoplasmic rRNA is taken as 0.71 × 10 6, as determined by sedimentation equilibrium, the molecular weights of the 12 s and 16 s components are 0.35 × 10 6 and 0.54 × 10 6, respectively. These molecular weight values are in good agreement with the relative values predicted from sedimentation velocity measurements, but not with the relative values based on gel electrophoresis.

Molecular weights of the ribosomal ribonucleic acid of fungi.

The molecular weights of the 18s and 25s ribosomal RNA components of fungi from all major classes were determined by electrophoresis in polyacrylamide gels. The molecular weight of the 18s RNA was found to be very similar for all fungi (range 0.71–0.75 million) and about 4–5% larger than the 18s RNA of HeLa cells and soybean. The molecular weight of the 25s RNA ranged between 1.45 million in the Myxomycetes and 1.30–1.31 million in the Ascomycetes and Basidiomycetes. The differences in the 25s RNA molecular weights between various classes of fungi were interpreted as being in agreement with a monophyletic origin of the Chytridiomycetes, Zygomycetes, Ascomycetes and Basidiomycetes, and independent origins for the Myxomycetes and the Oomycetes. The Hyphochytridiomycete examined could not be placed unequivocally in any group on the basis of its 25s RNA. Fungal RNA extracted with a p-aminosalicylate-triisopropylnaphthalene sulfonate-phenol mixture at 40–60°C contained a high molecular weight aggregate of the 18s and 25s ribosomal RNA; this suggested significant base sequence homology between the two ribosomal RNA species in fungi.

Variation with temperature of the apparent molecular weight of rat-liver mitochondrial RNA, determined by gel electrophoresis

The electrophoretic mobility of the two major RNA components of rat-liver mitochondria gradually decreases relative to the mobility of the cell-sap ribosomal RNA components when the temperature of electrophoresis is raised from 2 to 28°. Hence, the calculated apparent molecular weight of rat mitochondrial RNA is 40% higher at 28 than at 2°. This dependence of apparent molecular weight on the temperature of electrophoresis can explain the large differences in apparent molecular weight reported for the mitochondrial RNAs from different animals.

Size determination of mitochondrial ribosomal RNA from Aspergillus nidulans by electron microscopy

Mitochondrial ribosomal RNA from Aspergillus nidulans has been visualized in the electron miscroscope and compared to its homologous cytoplasmic ribosomal RNA and to Escherichia coli ribosomal RNA. The mean chain lengths of the two mitochondrial ribosomal RNA components are found to be 0.47 and 0.91 μ, whereas those of cytoplasmic ribosomal RNA are 0.52 and 1.10 μ and those of E. coli ribosomal RNA are 0.40 and 0.72 μ. Based upon its length, mitochondrial ribosomal RNA appears larger in size than bacterial-type ribosomal RNA but smaller than cytoplasmic ribosomal RNA.