Nucleolar function of the dense crescent in the yeast nucleus : A biochemical and ultrastructural study

A fraction rich in dense crescent material, as judged by electron microscopy, has been isolated from yeast nuclei. Double-labelling experiments with ¹⁴C-uracil and ³H-Me-methionine show that nearly all of the 37S and 28S ribosomal precursor RNA is confined to this dense crescent fraction. The presence of both 37S and 28S ribosomal precursors in the dense crescent fraction strongly suggests that both biosynthesis and processing of the ribosomal precursors take place in the dense crescent. The biochemical and electron microscopic data represent strong evidence that the dense crescent in yeast has to be regarded as a nucleolus in both morphological and biochemical sense. Apparently the yeast cell resembles the higher eukaryotes with regard to the organization within a nucleolus of the synthesis of ribosomal RNA via large precursor molecules.     

An excess of the small ribosomal subunits and a higher rate of turnover of the 60 S than of the 40 S ribosomal subunits in L cells grown in suspension culture.

A considerable excess of small ribosomal subunits was observed in L cells grown in suspension culture. The ratio between the small and large ribosomal subunits in the cytoplasm was estimated to be 1.17 ± 0.05 for cells dividing every 20 to 24 hours.The 60 S ribosomal subunits were turning over much faster than the 40 S subunits. Half-lives of 155 ± 20 hours for 18 S ribosomal RNA and 82 ± 15 hours for 28 S ribosomal RNA were observed under conditions where the cell number doubled every 24 hours and the viability was 95%. By correcting for cell death the half-lives of 18 S and 28 S ribosomal RNA were estimated to be approximately 300 hours and 110 hours, respectively. During storage of isolated ribosomes the small ribosomal subunits were degraded faster than the large subunits. This shows that the degradation of 60 S subunits was not an artifact taking place during the isolation procedure.It is postulated that the small ribosomal subunits are protected by protein to a greater extent than the 60 S subunits in these rapidly growing cells in suspension culture. The protection may take place both in the nucleus during synthesis, thus avoiding degradation (“wastage”) of nascent subunit precursors, and later in the cytoplasm. A calculation has been carried out to show that the observed excess of small subunits may be accounted for on the basis of a 1:1 synthesis of the small and large ribosomal subunits in the nucleus and different degradation rates in the cytoplasm. The results do not exclude the possibility of a difference in the “wastage” of 18 S and 28 S ribosomal RNA in the nucleus in addition to the difference in the turnover rates in the cytoplasm.     

Synthesis and intercellular transport of ribosomal RNA in the ovary of the milkweed bug Oncopeltus fasciatus

Analysis of cellular and subcellular fractions obtained by dissection and subsequent purification of the main components of the Oncopeltus fasciatus ovary reveals that the uptake and incorporation of tritiated uridine into high molecular weight RNA differs markedly between the trophic syncytium and the oöcytes. The pattern of labelling indicates that the trophic syncytium is actively engaged in the synthesis of ribosomal RNA, but that the oöcytes do not synthesize detectable amounts of ribosomes. The ribosomal RNA which eventually appears in the cytoplasm of the oöcytes is postulated to come from the trophic syncytium via the nutritive cords. There is no other fraction of non-4 S RNA detectable in the oöcytes by these methods.     

Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins

Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.     

Ribosomes and Polysomes in Cucumber Leaves during Growth and Senescence

The quantity of RNA in the ribosomal fraction of the first leaf of cucumber (Cucumis sativus) increases during growth, reaches a maximum before the final fresh weight is attained, and then decreases. The main changes are in the free ribosome fraction, the quantity of membrane-bound ribosomes remaining about constant. Few 65.5S chloroplast ribosomes are present in small leaves; however, they increase in quantity rapidly during growth and form about half of the ribosomes present in the mature fully green leaf. The cytoplasmic ribosomes have a sedimentation coefficient of 77.6S. Ribonuclease-sensitive polysomes were present in leaves of all ages except possibly the very oldest. The proportion of ribosomes in polysome form decreases during growth and then remains roughly constant during senescence. Following maturation of the leaf, the rate of incorporation of ³²P into ribosomal-fraction RNA begins to decline. This decline could account for the loss of ribosomes during the early stages of senescence. The possibility that leaf ribonuclease might be responsible for the final, more rapid loss of RNA, is discussed.     

Kern-Plasma-Transport neusynthetisierter rRNS in Zellen einer Suspensionskultur von Petroselinum sativum

Summary A rapidly labelled rRNA precursor can be detected in callus cells of Petroselinum sativum grown on a liquid synthetic medium. Its molecular weight has been calculated to be 2.3×10⁶. This value agrees with that of the rRNA precursor from other plant material. In order to follow the synthesis and processing of rRNA in time and to correlate single steps in this process with cell organelles it was necessary to obtain pure fractions of nuclei and ribosomes. The isolation method for nuclei is given in detail. The nucleic acids are separated on polyacrylamide gels of low acrylamide concentration. Pulse-chase experiments show that the rRNA precursor is split into two fragments within the nucleus: an 18S and a 25S component. The 18S RNA leaves the nucleus rapidly. It is already found quantitatively in the ribosomal fraction after 30–60 min chase. At that time the 25S RNA is still within the nucleus; it appears much later in the ribosomes. Since the increase in ribosomal label occurs simultaneously with the decrease in nuclear label, it is concluded that there is no degradation of 18S RNA within the nucleus. Apparently there are two distinct transport mechanisms with different kinetics for the two RNA components.     

Rates of synthesis of ribosomal protein and total ribonucleic acid through the cell cycle of the fission yeast Schizosaccharomyces pombe

The rates of synthesis of ribosomal proteins through the cell cycle of the fission yeast Schizosaccharomyces pombe have been examined by spec. act. estimations of isolated 80S ribosomes pulse-labelled with ³⁵S-sulphate. The spec. act. have minimum values at the beginning (0.0) and maxima between 0.6 and 0.9 of the cell cycle. This pattern in spec. act. is also shown by isolated 80S ribosomes pulse-labelled with ³H-uridine during synchronous cultures and is in marked contrast to the small, random variations in the spec. act. of isolated 80S ribosomes from control, asynchronous cultures pulse-labelled with ³⁵S-sulphate or ³H-uridine.A detailed examination of the rates of synthesis of total RNA through the cell cycle measured by the rates of incorporation of ³H-uridine and ³H-adenine shows a step in the rates of incorporation at the time of DNA synthesis. This step has further been shown to be independent both of the uridine concentration, over a range from 0.03 μM to 820 μM, and of pre-filling the adenine pool. This step thus appears to be independent of variations in rates of uptake of both purines and pyrimidines, or fluctuations in the pool size of the precursors and may be explained as a gene-dosage effect.The step in the pattern of synthesis of total RNA has been shown to yield a cyclic pattern in the spec. act. of the total RNA through the cell cycle. This pattern is similar to that of the spec, act. of RNA and of protein recovered from ribosomes. The variation exhibited by the ribosomal proteins is believed to be a consequence of the step in the pattern of RNA synthesis, with a concomitant fluctuation in the pool of ribosomal proteins synthesised continuously through the cell cycle.     

RNA metabolsim during vegetative growth and morphogenesis of the cellular slime mold Dictyostelium discoideum

During vegetative growth of the cellular slime mold Dictyostelium discoideum, RNA is rapidly labeled by radioactive precursor and both the 25 S and the 17 S ribosomal RNA species appear in the cytoplasm 6–7 min after the onset of labeling. Thirty minutes after further incorporation of radioactive RNA precursors has been blocked, less than 10% of the label in RNA is associated with the nuclear fraction. After aggregation of the slime mold amoebae, RNA appears in the cytoplasm at a reduced rate, the small ribosomal subunit appearing in the cytoplasmic fraction more slowly than the larger ribosomal subunit. Some labeled RNA remains in the nuclei of developing cells long after the incorporation of ³H-uridine is blocked.     

Mitochondrial ribosome assembly in Neurospora crassa. Chloramphenicol inhibits the maturation of small ribosomal subunits.

Recent results suggest that, in Neurospora crassa, one small subunit mitochondrial ribosomal protein (S-4a, M_r 52,000) is synthesized intramitochondrially (Lambowitz et al., 1976). We now find that, when wild-type cells are treated with chloramphenicol to block mitochondrial protein synthesis, the maturation of 30 S mitochondrial ribosomal subunits is rapidly inhibited and there is an accumulation of CAP-30 S particles which sediment slightly behind mature small subunits. Electrophoretic analysis suggests that the CAP-30 S particles are deficient in several proteins including S-4a and that they are enriched in a precursor RNA species that is slightly longer than 19 S RNA. Chloramphenicol also appears to inhibit the maturation of 50 S ribosomal subunits, but this effect is much less pronounced. Continued incubation in chloramphenicol leads to a decrease in the proportion of total mitochondrial ribonucleoprotein present as monomers, possibly reflecting the depletion of competent subunits. After long-term (17 h) growth in chloramphenicol, mitochondrial ribosome profiles from wild-type cells show decreased ratios of small to large subunits, a feature which is also characteristic of the poky (mi-1) mutant. Pulse-labeling experiments combined with electrophoretic analysis show that the synthesis of mitochondrial ribosomal RNAs is relatively unaffected by chloramphenicol and that, despite the deficiency of small subunits, 19 S and 25 S RNA are present in normal ratios in whole mitochondria. By contrast, 19 S RNA in poky mitochondria is rapidly degraded leading to a decreased ratio of 19 S to 25 S RNA. The significance of these results with respect to the etiology of the poky mutation is discussed and a model of mitochondrial ribosome assembly that incorporates all available data is presented.     

Ribosomes of Brachionus plicatilis (Rotifera) – distribution in homogenate fractions and general properties

(1) The content of DNA, RNA and of proteins of Brachionus plicatilis was estimated and the distribution of RNA and of proteins of different homogenate fractions characterised. (2) Ribosomes were isolated from Brachionus plicatilis homogenates and were characterised by gradient centrifugation. (3) Unlike the RNA content, the yield of ribosomes from different homogenate fractions is strongly dependent on the concentration of Mg²⁺-ions in the buffers. Likewise resuspension of ribosomes is more effective in Mg²⁺- (or Ca²⁺-) free buffers. (4) Dissociation of ribosomes was brought about by centrifugation of ribosomes in gradients containing less than 4 mM Mg²⁺. In this case, beside the peaks of subunits, a peak in the region of 80 S remained which vanished only under conditions destroying ribosomal material altogether. (5) Proteins were isolated from ribosomal subunits and from undissociated ribosomes and were characterised by two-dimensional gel electrophoresis techniques. Patterns of 51 spots were regularly obtained from large subunits and patterns of 41 spots from small subunits. The undissociated ribosomes showed 83 spots, most of which could be attributed to the large or the small subunit. The ribosomal proteins have molecular masses of between 11000 and 56000 Da, while the molecular mass of the total protein content of Brachionus ribosomes was estimated to be 1.8 ±0.5) ×10⁶ Da.     

Decomposition of ribosomal particles in Escherichia coli treated with mitomycin C

Exposure of cells of Escherichia coli to mitomycin C (5 mug/ml) resulted in a marked change in the sedimentation profiles of the cell-free extracts, indicating a specific decomposition of ribosomal particles. When the extracts were prepared in the presence of 0.01 m Mg(++) and analyzed by sucrose density gradient centrifugations, the 100S fraction disappeared rapidly from the treated cells. The 70S ribosomes were also degraded, but more slowly, with a concomitant accumulation of a fraction having a sedimentation coefficient of about 50S. However, decomposition of the 70S ribosomes was preceded by an almost complete loss of the 50S ribosomal subunits, as revealed by sedimentation analyses in the presence of 10(-4)m Mg(++). Synthesis of the ribosomes in the treated cells was also suppressed, being demonstrated by a lower incorporation of uracil-2-(14)C into the ribosomal fractions. However, the change in the ribosomal profile in the treated cells apparently resulted from the decomposition of pre-existing ribosomes, rather than from the inhibition of the net synthesis of ribosomes. Sedimentation analyses and chromatography of the nucleic acids extracted from the treated cells indicated extensive but delayed degradation of the ribosomal ribonucleic acid (RNA), but not of the soluble RNA or deoxyribonucleic acid fractions. Altered structure of the ribosomes in the treated cells was also indicated by their lower melting temperature, broadened thermal profile, higher electrophoretic mobility, and extreme sensitivity to ribonuclease treatment, compared with normal ribosomes. The synthesis of messenger RNA was inhibited progressively with time in the treated cells.     

Evidence that glyoxysomal malate synthase is segregated by the endoplasmic reticulum

At the onset of castor bean (Ricinus communis) germination, 76% of the cellular malate synthase activity of the endosperm tissue was located in the microsomal fraction, with the remainder in the glyoxysomal fraction. During later developmental stages, when rapid malate synthase synthesis was occurring, an increasing proportion of the enzyme was recovered in glyoxysomes. The kinetics of [³⁵S]methionine incorporation into microsomal and glyoxysomal malate synthase in 2-day-old endosperm tissue was followed by employing antiserum raised against glyoxysomal malate synthase to precipitate specifically the enzyme from KCl extracts of these organelle fractions. This experiment showed that microsomal malate synthase was labeled before the glyoxysomal enzyme. When such kinetic experiments were interrupted by the addition of an excess of unlabeled methionine, ³⁵S-labeled malate synthase was rapidly lost from the microsomal fraction and was quantitatively recovered in the glyoxysomal fraction.

Free cytoplasmic ribosomes were separated from bound ribosomes (rough microsomes) using endosperm tissue labeled with [³⁵S]methionine or ¹⁴C-amino-acids. Nascent polypeptide chains were released from polysome fractions using a puromycin-high salt treatment, and radioactive malate synthase was shown to be exclusively associated with bound polysomes.

Together these data establish that malate synthase is synthesized on bound ribosomes and vectorially discharged into the endoplasmic reticulum cisternae prior to its ultimate sequestration in glyoxysomes.

     

Control of protein synthesis in brine shrimp embryos by repression of ribosomal activity

Undeveloped encysted embryos of the brine shrimp, Artemia salina, contain a large quantity of metabolically repressed 80S ribosomes. These ribosomes appear to be inactive or nonfunctional due to the presence of an inhibitor protein on their 60S subunit. During development the inhibitor is released or inactivated and the 80S ribosomes and their constituent subunits become fully functional in a poly(U)-directed protein-synthesizing system. The inefficiency of most 80S ribosomes from undeveloped Artemia embryos appears to be due to their inability to form stable complexes with poly(U) and phe-tRNA in the presence of elongation factor, EF-1. A potent inhibitor of protein synthesis has also been found in the 105,000g supernatant fraction from undeveloped Artemia embryos. The exact nature of this inhibitor has not been ascertained but it appears to be a heat-labile protein devoid of RNase and protease activity. It is not known whether this inhibitor is the same as that associated with 60S ribosomal subunits of undeveloped cyst ribosomes.     

Binding and translation of turnip-yellow-mosaic-virus ribonucleic acid by skeletal-muscle ribosomes from normal and diabetic rats

Ribosomes from skeletal muscle of diabetic rats were less active than normal ribosomes in protein synthesis directed by turnip-yellow-mosaic-virus RNA. The proportion of ribosomes from muscle of diabetic rats capable of binding turnip-yellow-mosaic-virus RNA was greater than normal, but there was no difference in the equilibrium constants for the binding reaction. The turnip-yellow-mosaic-virus RNA was bound preferentially to the small (40S) ribosomal subunit, whereas the decrease due to diabetes in its translation was associated with the large (60S) subunit. Thus the diminished capacity of ribosomes from muscle of diabetic rats to translate turnip-yellow-mosaic-virus RNA was not the result of decreased binding of the template.     

Nuclear export competence of pre-40S subunits in fission yeast requires the ribosomal protein Rps2

Ribosome biogenesis is an evolutionarily conserved pathway that requires ribosomal and nonribosomal proteins. Here, we investigated the role of the ribosomal protein S2 (Rps2) in fission yeast ribosome synthesis. As for many budding yeast ribosomal proteins, Rps2 was essential for cell viability in fission yeast and the genetic depletion of Rps2 caused a complete inhibition of 40S ribosomal subunit production. The pattern of pre-rRNA processing upon depletion of Rps2 revealed a reduction of 27SA₂ pre-rRNAs and the concomitant production of 21S rRNA precursors, consistent with a role for Rps2 in efficient cleavage at site A₂ within the 32S pre-rRNA. Importantly, kinetics of pre-rRNA accumulation as determined by rRNA pulse-chases assays indicated that a small fraction of 35S precursors matured into 20S-containing particles, suggesting that most 40S precursors were rapidly degraded in the absence of Rps2. Analysis of steady-state RNA levels revealed that some pre-40S particles were produced in Rps2-depleted cells, but that these precursors were retained in the nucleolus. Our findings suggest a role for Rps2 in a mechanism that monitors pre-40S export competence.     

Study of the internal structure of Escherichia coli ribosomes by neutron and x-ray scattering.

Neutron scattering curves of the small and large subparticles of Escherichia coli ribosomes are presented over a wide range of scattering angles and for several contrasts. It was verified that the native ribosome structure was not affected by ²H₂O in the buffer. The reliability of the neutron scattering curves, obtained in H₂O buffer, was established by X-ray scattering experiments on the same material.The non-homogeneous distribution of RNA and protein in the subparticles of E. coli ribosomes is confirmed, with RNA predominantly within the particle and protein predominantly on its periphery. The distances between the centres of gravity of the RNA and protein components do not exceed 25 Å and 30 Å, in the large and small subparticles, respectively.The volume occupied by the RNA within the large and small subparticles is determined. The ratio of the “dry” volume of the RNA to the occupied volume is found to be 0.56; it is the same in both subparticles. Such packing of RNA is characteristic of single helices of ribosomal RNA at their crystallization and of the helices in transfer RNA crystals. A conclusion is drawn that RNA in ribosomes is in a similar state.Experimental scattering curves for the small subparticle depend significantly on the contrast in the angular region in which the scattering is mainly determined by the particle shape. The scattering curve, as infinite contrast is approached, is similar to that calculated for the particle as observed by electron microscopy. Thus, the long-existing contradiction between electron microscopy data (an elonggated particle with an axial ratio 2:1) and X-ray data (an oblate particle with an axial ratio 1:3.5), concerning the overall shape of the 30 S subparticle, is settled in favour of electron microscopy. The experimental neutron scattering curve of RNA within the small subparticle is well-described by the V-like RNA model proposed recently by Vasiliev et al. (1978).Experimental data are given to support the hypothesis that the maxima in the X-ray scattering curves, in the region of scattering angles corresponding to Bragg distances of 90 to 20 Å, arise from the ribosomal RNA component alone. It is shown that the prominence of the peaks in this region of the scattering curve depends only on the scattering fraction of the RNA component. The scattering fraction can be changed both by using the “native contrast” (ribosomal particles containing different amounts of protein) and by varying the solvent composition. The maxima are most pronounced where the RNA scattering fraction is highest or in solvents where the protein density is matched by the solvent. The scattering vectors of the maxima in the X-ray and neutron scattering curves, however, remain unchanged. This allows us to propose the tight packing of RNA as a common principle for the structural arrangement of RNA in ribosomes.     

Small protein B interacts with the large and the small subunits of a stalled ribosome during trans-translation

During trans-translation, stalled bacterial ribosomes are rescued by small protein B (SmpB) and by transfer-messenger RNA (tmRNA). Stalled ribosomes switch translation from the defective messages to a short internal reading frame on tmRNA that tags the nascent peptide chain for degradation and recycles the ribosomes. We present evidences that SmpB binds the large and small ribosomal subunits in vivo and in vitro. The binding between SmpB and the ribosomal subunits is very tight, with a dissociation constant of 1.7 × 10⁻¹⁰ M, similar to its K_D for the 70S ribosome or for tmRNA. tmRNA displaces SmpB from its 50S binding but not from the 30S. In vivo, SmpB is detected on the 50S when trans-translation is impaired by lacking tmRNA or a functional SmpB. SmpB contacts the large subunit transiently and early during the trans-translational process. The affinity of SmpB for the two ribosomal subunits is modulated by tmRNA in the course of trans-translation. It is the first example of two copies of the same protein interacting with two different functional sites of the ribosomes.     

Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-³H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNA's. Both mature ribosomal RNA's compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNA's. The large molecular weight precursor (32 S) of both ribosomal RNA's appears to be 2.4 × 10⁶ daltons in size. Maturation to large subunit RNA (1.28 × 10⁶ daltons) is assumed to involve an intermediate ~1.6 × 10⁶ daltons in size, while cleavage to form small subunit RNA (0.72 × 10⁶ daltons) presumably involves a 0.9 × 10⁶ dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNA's, the mitochondrial precursor RNA has a strikingly low G + C content.     

A small, unstable RNA molecule of Escherichia coli: spot 42 RNA. II. Accumulation and distribution.

We have studied the accumulation and distribution of a small, unstable RNA molecule of Escherichia coli called spot 42 RNA (Ikemura &; Dahlberg, 1973b). This molecule has sequence and structural features characteristic of messenger RNAs and leader RNAs (Sahagan &; Dahlberg, 1979). It is present in 100 to 200 copies per cell when cells are grown in media supplemented with glucose. Although it accumulates in cells that have been deprived of an essential amino acid or phosphate or ammonium ions, the amount of spot 42 RNA is greatly reduced when cells are grown on non-glucose carbon sources or when cyclic AMP is added to a glucose-grown culture. Thus accumulation of spot 42 RNA is inversely proportional to the intracellular concentration of cAMP.About half of the spot 42 RNA in a cell is isolated in association with bacterial nucleoids. It is the only 4 to 6 S RNA which is enriched in this fraction. This molecule also cofractionates with 5 S ribosomal RNA during the preparation of ribosomes and ribosome fractions.The possibility that spot 42 RNA is a leader sequence whose level of accumulation is modulated by the intracellular level of cAMP is discussed.