Ribosomal RNA Components Research Articles

Disaggregation of giant “DNA-like RNA” of yeast by denaturation in the presence of formaldehyde

In yeast, cycloheximide causes the accumulation of heterogeneous RNA, most of which is high molecular weight RNA sedimenting faster than the 26E ribosomal RNA component, which has a molecular weight of 1.3 × 10 6. Heating of this heterogeneous RNA in the presence of formaldehyde shows that this RNA is an aggregate of molecules tha t have molecular weights ranging from about 0.2 × 10 6 to 1.4 × 10 6.

The formation of ribonucleic acid in yeast: Hybridization of high molecular weight RNA species to yeast DNA

Hybridization in solution was used to investigate the sequence homology between DNA and several high molecular weight species of Saccharomyces carlsbergensis. In agreement with others, about 1.3 and 0.7% of the DNA was found to be homologous respectively to the 26S and 17S ribosomal RNA components. Yeast ribosomal RNA is transcribed from a satellite with a buoyant density slightly higher (1.703) than main yeast DNA (1.698). The experiments indicate that the satellite contains some DNA other than that homologous to ribosomal RNA. Other experiments showed that the RNA synthesized in the presence of cycloheximide is a transcript of at least 20% of the genome.

Isolierung und Charakterisierung schnell markierter, hochmolekularer RNS aus frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

By culturing of callus tissue originating from root explants of Petroselinum sativum in a synthetic liquid medium under aeration, freely suspended single cells and small clusters consisting of mostly five cells were obtained. The rapidly dividing cells did not exhibit any morphogenesis. Their nucleic acid metabolism was investigated by pulse experiments with 32P-orthophosphate. Rapidly labelled RNA was prominently found associated with high molecular RNA. During the fractionation of the total nucleic acids on MAK columns it was eluted after the ribosomal RNA components. Its base ratio, however, differed from the latter in that the AMP content was higher than the GMP content. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis resulted in the separation of the ribosomal RNA from the rapidly labelled RNA, thus proving the higher molecular weight of the latter. Based upon the migration in the gel a sedimentation coefficient of approximately 32S was calculated. The possible function of the heavy rapidly labelled RNA component as precursor of ribosomal RNA is discussed.

Structural comparison of 26S and 17S ribosomal RNA of yeast.

THE two large ribosomal RNA components (26S and 17S rRNA) of yeast exhibit a noteworthy structural similarity as judged by nucleotide composition1, the degree of methylation2, and the extensive cross hybridization with homologous DNA3. Because the 17S rRNA can compete with much more than 50 per cent of 26S rRNA in hybridization with homologous DNA3, and because the estimated chain length of the 26S rRNA is about twice that of 17S rRNA (ref. 4 and R. C. v. d. B., J. Retel and R. J. P., manuscript in preparation), it seems reasonable to assume that the 26S rRNA comprises two segments, each showing some similarity to a 17S rRNA molecule. But although competition in hybridization with DNA does mean that the two RNA species exhibit some primary structural homology, it certainly does not indicate that they have identical base sequences.

Isolation and characterization of Euglena gracilis cytoplasmic and chloroplast ribosomes and their ribosomal RNA components

1. 1. Euglena gracilis cytoplasmic and chloroplast polysomes have been isolated and characterized by their s-values relative to rat-liver polysomes and Escherichia coli ribosomes. 2. 2. Euglena contains 87-S ribosomes in the cytoplasm and 68-S ribosomes in the chloroplasts. 3. 3. The Euglena cytoplasmic ribosome contains two rRNA components sedimenting as 24-S and 20-S molecules, while the chloroplast ribosome is composed of two slightly smaller rRNA molecules, 22 S and 17 S. The four rRNA molecules are sufficiently distinct to be separable in a sucrose gradient. 4. 4. When compared with other eucaryotic cytoplasmic ribosomes the Euglena cytoplasmic ribosome is unique in overall s values and RNA composition. The Euglena chloroplast ribosome is similar to other 70-S type ribosomes in both size and RNA content. 5. 5. These results are in contradiction to earlier reports regarding Euglena cytoplasmic and chloroplast ribosomes and their respective rRNA components.

Effects of ionic conditions on the structure of ribosomal RNA components

The hydrodynamic shape, conformation, and thermal stability of ribosomal 23S RNA have been studied by sedimentation velocity analysis, circular dichroism, ultraviolet absorption, and thermal denaturation. The results show that magnesium ion is critically required for maintaining the structural integrity of ribosomal 23S RNA. Upon removal of magnesium ions, the sedimentation coefficient of 21.1 S for native 23 S RNA is reduced to approximately 8.5 S, indicative of a large unfolding of the RNA. A large increase in absorbance at 260 nm in UV spectra of 23S RNA and a decrease in ellipticity near the 265 nm peak which was followed by parallel changes in the 237 nm trough and the 208 nm trough. Furthermore, the melting temperature (Tm) of the 23S RNA is lowered to 50°C from 65°C with a concomitant decrease in the cooperativity of the melting curve of 23S RNA.

Non-random variability in evolution of base compositions of ribosomal RNA.

DURING the past few years, base composition data have accumulated in the literature for the two chief components of ribosomal RNA (rRNA): the larger (23–28S RNA) and the smaller (16–18S RNA). I wish to draw attention to some peculiarities in these data.

Einbau von 3H-Uracil in die RNA steriler Anzuchten von Utricularia stellaris mit und ohne Rohrzucker

Sucrose has been shown to increase the growth of Utricularia stellaris in axenic culture (Harder and Zemlin, 1967). Comparable cultures of Utricularia were incubated for 3 and 7 hours with tritiated uracil, and then nucleic acids were prepared. With sucrose, the incorporation is about 5 times higher than that with the inorganic salt cultures. After chromatographic separation of the nucleic acids on MAK-columns, different peaks of short time labelled RNA with high specific activity were found. These peaks indicate the presence of two low molecular weight RNA components in the inorganic culture. These peaks also have been found in cultures with sucrose, but in these cultures there is another predominant peak of high molecular weight RNA which is eluted from the column after the greater ribosomal RNA component.

Effects of cycloheximide and 5-fluorouracil on the synthesis of ribonucleic acid in yeast

A fractionation by means of phenol treatment at different temperatures revealed that in yeast in the presence of cycloheximide small amounts of ribosomal 28-S RNA are synthesized. Virtually no formation of ribosomal 18-S RNA occurs. In the presence of 5-fluorouracil both ribosomal RNA components are found to be made. A study of the methylation of RNA formed in the presence of cycloheximide and 5-fluorouracil confirmed these observations but suggested also that small amounts of ribosomal precursor RNA accumulate. A closer investigation of the fate of this precursor RNA synthesized in the presence of cycloheximide revealed that this RNA was utilized mainly for the formation of the ribosomal 18-S species, indicating a selective accumulation of a precursor RNA for this ribosomal RNA component by the antibiotic. The remainder of the RNA accumulating in the presence of the antibiotic is also converted into ribosomal RNA. 5-Fluorouracil was found to inhibit this latter conversion. The observations are discussed in view of current hypotheses with regard to the formation of ribosomal RNA.

The effect of light and inhibitors on chloroplast and cytoplasmic RNA synthesis.

Chloroplast RNA is synthesized in dark-grown radish cotyledons at about one-third the rate of that in the light. The synthesis, however, continues for longer in the dark and the percentage of chloroplast RNA can approach that in light-grown tissue. Light stimulates the synthesis and accumulation of both cytoplasmic and chloroplast RNA, but shows a 4-fold greater stimulation of the chloroplast RNA. Chloramphenicol, streptomycin and cycloheximide inhibit the synthesis of chloroplast RNA with little effect on cytoplasmic RNA. 5-Fluorouracil inhibits the synthesis of cytoplasmic more than chloroplast RNA. Synthesis of the 0.56 x 10(6) mol wt chloroplast RNA is inhibited much less than the other ribosomal RNA components by actinomycin D.

Sedimentation studies on RNA from proplastids of Zea mays

The sedimentation rate of ribosomal RNA from purified proplastids of dark-grown leaves of Zea mays was compared with the sedimentation rate of cytoplasmic ribosomal RNA in whole leaf extracts by use of high-resolution sucrose gradients. Both components of plastid ribosomal RNA can be resolved from cytoplasmic ribosomal RNA when mixtures of RNA from both sources are cosedimented on the same gradient. Approximate sedimentation coefficients were determined by comparing the rate of sedimentation of the plant RNA with 32P-labeled RNA extracted from Escherichia coli. Values of 15 S and 23 S were obtained for proplastid RNA; values of 16 S and 26 S were obtained for cytoplasmic RNA. In these preparations, it appears that the smaller proplastid ribosomal component can be distinguished from 16 S bacterial ribosomal RNA.

Function of mitochondrial DNA in yeast.

MITOCHONDRIA contain small amounts of DNA which differ in several chemical and physical properties from nuclear DNA (reviewed in ref. 1). Mitochondrial DNA (MDNA) of yeast2–6 is replicated by a mitochondrial DNA polymerase7 which is different from the nuclear enzyme. (unpublished results of U. Wintersberger and E. W.). A genetic function of MDNA is indicated because it serves as a template for a mitochondrial RNA polymerase8,9. Mitochondrial RNA (MRNA) has been isolated and characterized using highly purified yeast mitochondria. MRNA of yeast separates into three species when subjected to sucrose gradient centrifugation, one with a sedimentation coefficient of about 4S, the other two sedimenting faster. The 4S RNA accepts amino-acids in a manner similar to cytoplasmic tRNA; the other two RNA components were assumed to be derived from mitochondrial ribosomes9. The two ribosomal RNA (rRNA) components of the mitochondria differ in their sedimentation properties from 25S and 17S RNA of the cytoplasmic 80S ribosomes of yeast, and, in contrast, closely resemble the 23S and 16S RNA species of bacterial 70S ribosomes10,11. Hybridization experiments show that yeast MRNA hybridizes readily with MDNA, in sharp contrast to cytoplasmic rRNA which forms hybrids with MDNA very poorly but anneals extensively to nuclear DNA11,12.

Characterization of ribosomes of Tetrahymena pyriformis

Some of the properties of the 80-S ribosome of Tetrahymena pyriformis have been examined. We estimate from sedimentation coefficient (79.5-S) and intrinsic viscosity (0.074 dl/g) a mol. wt. of 4.6·10 6 for the ribonucleoprotein particles. The RNA and protein ratio of the ribosomes is close to one. The high molecular weight ribosomal RNA (rRNA) components sediment at 26-S and 14-S. Disc-gel electrophoresis runs at pH 3.8 show 13 prominent bands while little, if any, protein migrated into the gel when the run was made at pH 9.4. The molar ratio of polyamine-nitrogen to RNA-phosphorus for these ribosomes is 0.24. Spermidine and putrescine are present at about the same level and account for 95 % of the polyamines bound to the ribosome.

Studies on microbial RNA: V. A comparison of the in vivo methylated components of ribosomal RNA from Escherichia coli and Saccharomyces cerevisiae

1. Methylated bases in ribosomal RNA (rRNA) were estimated by a method consisting of (a) in vivo methyl labelling of the rRNA, (b) isolation of the rRNA, (c) acid hydrolysis of the RNA in the presence of carriers, (d) separation of the hydrolysis mixture by two-dimensional chromatography and (e) estimation of the radioactivity in the separated spots. Ribose-methylated nucleotides were estimated separately. 2. The procedure was used to compare five strains of Escherichia coli and two strains of Saccharomyces cerevisiae. The two organisms differ markedly in the distribution of methylated compounds in their rRNA, while strains of the same organism are very similar. The content of O- ribose methyl groups was high in the yeast strains but rather low in the bacteria.

Studies of the chain termini and alkali-stable dinucleotide sequences in 16 s and 28 s ribosomal RNA from L Cells

The terminal groups of the polynucleotide chains in each of the 16 s and 28 s components of ribosomal RNA from L cells were investigated using an alkali hydrolysis technique. The results of the study indicate that the polynucleotide chains in each component can be considered to have a formal structure based on a repeating 5′-mononucleotide unit, i.e. (pN) n , or (3′-linked end) pNpNpN .......... pNpNpN (5′-linked end) since alkali hydrolysis released nearly equimolar amounts of nucleoside 2′(3′), 5′-diphosphates (from 3′-linked termini) and nucleosides (from 5′-linked termini). In the case of 16 s RNA, the principal nucleoside derived from 5′-linked termini was adenosine, and the principal nucleoside diphosphate derived from 3′-linked termini was uridine 2′(3′),5′-diphosphate, i.e. pUpNpN .......... pNpNpA, but, owing to heterogeneity at both chain ends, it is not possible to assess what proportion of the total polynucleotides possess both of the principal end groups in the same chain. In the case of 28 s RNA, the principal nucleoside derived from 5′-linked termini was uridine, and the principal nucleoside diphosphate derived from 3′-linked termini was cytidine 2′(3′),5′-diphosphate, i.e. pCpNpN .......... pNpNpU, but, again, owing to heterogeneity at both chain ends, it is not possible to assess what proportion of the total polynucleotides possess both of the principal end groups in the same chain. On the basis of analyses for 5′-linked termini, the mean chain length for 16 s RNA was 1.4 × 10 3, and the mean chain length of 28 s RNA was 2.0 × 10 3 nucleotide residues. Preliminary results from methodologically allied studies of alkali-stable, 2′- O-methyl ribose-containing dinucleotide sequences showed that most of the 16 possible sequences were present in each of the 16 s and 28 s components of L cell ribosomal RNA. These alkali-stable dinucleotide sequences account for about 2 mole % of the constituent nucleotide residues in each of the 16 s and 28s components.

The effect of thioacetamide on the maturation of high-molecular-weight ribonucleic acid in tumour cells.

1. Although thioacetamide treatment of Krebs II ascites-tumour cells did not markedly affect the rate of RNA synthesis in vivo, it caused the formation of an unusual single-stranded RNA component sedimenting at approx. 26s. 2. The maturation process leading to the formation of methylated RNA was examined by following the kinetics of incorporation into RNA of radioactivity from [G-(3)H]uridine and l-[Me-(14)C]methionine. In treated and untreated tumour cells extensive methylation was observed, not only of the ribosomal RNA species, but also of their precursors, especially the precursor species sedimenting at 35s. 3. Evidence is also presented to suggest that methylation of low-molecular-weight RNA species occurs both in the nucleus and in the cytoplasm of these tumour cells. 4. Thioacetamide did not appear to have an effect on RNA methylation in vivo, and in thioacetamide-treated cells the 26s RNA accumulated within the nucleus, where it was methylated. 5. It is postulated that the 26s RNA is most likely to arise as a result of a fault in the scission process that gives rise to the ribosomal RNA components from their high-molecular-weight precursors.

The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis.

1. Gels were prepared with recrystallized acrylamide and bisacrylamide. Electrophoresis was in tris-sodium acetate-EDTA buffer for 0.5 to 3hr. Gels were scanned at 280 or 265mmu. Techniques are described for slicing and radioactive counting. 2. The mobility of RNA was inversely related to the sedimentation coefficient and varied with gel concentration. Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation. It shows the two ribosomal RNA components, the 45s precursor, transfer RNA and minor components. In 5% and 7.5% gels, 4s and 5s RNA separated and ribosomal RNA was excluded. 3. The resolution is greater and more detailed than by centrifugation, and many samples can be analysed simultaneously and rapidly.

The nature of 7 s RNA in mammalian cells

An RNA component of ribosomes, distinct from transfer RNA, has been isolated from mouse liver and Sarcoma 180 cells. This RNA resembles transfer RNA in base composition but has a sedimentation coefficient of 7 s and does not bind amino acids. The RNA contains only trace amounts of pseudouridylic acid and does not contain methylated bases. Kinetic studies suggest that 7 s RNA production is associated with messenger RNA synthesis.

Incorporation of labelled precursors into the electrophoretic fractions of rat-liver ribonucleic acid.

1. Incorporation of [(32)P]orthophosphate and of [2-(14)C]orotic acid into rat-liver RNA was studied by agar-gel electrophoresis by using u.v.-densitometry and radioautography of dried agar electrophoretograms. 2. During the electrophoresis some low-molecular-weight contaminants, including inorganic phosphate present in the RNA preparations, were separated from the RNA fractions. Since nucleoside mono-, di- and tri-phosphates still interfered, the RNA preparations had to be subjected to a purification procedure [Sephadex G-25 or Dowex 1 (X8)]. 3. In RNA extracted from cytoplasm, isolated microsomes or ribosomes, whatever variations were made in the phenol procedure no special rapidly labelled RNA fraction was detected other than ;soluble' RNA and the ribosomal RNA components. 4. When the whole homogenate or cytoplasmic fraction was treated only with phenol (pH6) a considerable part of the cytoplasmic RNA was not extracted. The treatment of the cytoplasmic fraction with sodium dodecyl sulphate before the addition of phenol increased the yield of the high-molecular-weight RNA and at the same time a higher specific activity was found for the faster ribosomal RNA component. 5. The presence of four distinct rapidly labelled RNA fractions was established in the RNA not extracted by phenol, and they moved slower than the ribosomal RNA. They were extracted only with the use of phenol-sodium dodecyl sulphate at an elevated temperature.

Base composition of the newly synthesized RNA in the two ribosomal RNA components of rat liver

RNA isolated from the 600 x g supernatant of rat liver homogenate by means of a cold phenol-SDS procedure was fractionated by a preparative agar gel electrophoretic technique. The base composition of the two ribosomal RNA components and of s-RNA was determined from the UV-absorbancy data, as well as from the radioactivity of the four 2′(3′)-mononucleotides after different labeling times with 32p. The two ribosomal RNA components had statistically significant differences in their base composition, the lighter component showing a lower G and C content. The base composition position of the newly synthesized RNA differed from that of the total RNA in both ribosomal RNA components, having a lower G+ C a+ U ratio. This ratio approached that of DNA at 1 hr, and was close to that of ribosomal RNA at 4.5 hr labelling time. The conclusion is drawn that in rat liver cytoplasm a particular RNA fraction is present, which shows: a/ higher rate of turn-over; b/ electrophoretic mobility in agar gel exactly coinciding with that of the corresponding peaks of the ribosomal RNA, and c/ base composition significantly different from that of the ribosomal RNA.