Monomeric Ribosomes Research Articles

Phosphorylation of ribosomal protein S6 in the Ehrlich ascites tumor cell: Lack of effect of phosphorylation upon ribosomal function in vitro

The incorporation of 32P i into the ribosomal protein S6, which is a component of the 40 S ribosomal subunit, was measured in Ehrlich ascites tumor cells growing in suspension culture. During a 2-h incubation period, incorporation was 10-fold greater into S6 of cells growing exponentially in complete medium than in cells in which protein synthesis and growth were inhibited by omission of glutamine from the medium. Since the labeling of other phosphoproteins was not similarly depressed in glutamine-deprived cells, the decreased labeling most likely reflects a specific decrease in phosphate incorporation into S6 rather than a lower phosphate pool specific activity. The cycling of ribosomes is inhibited in glutamine-deprived cells, with an accumulation of monomeric ribosomes indicating an inhibition of protein synthesis initiation. To assess whether the decreased phosphorylation of S6 in the ribosomes of glutamine-deprived cells was responsible for their decreased ability to initiate protein synthesis and enter polyribosomes, a mixture of 14C-labeled ribosomes from rapidly growing cells and 3H-labeled ribosomes from glutamine-deprived cells was added to a rabbit reticulocyte lysate cell-free protein-synthesizing system in which ribosomes cycle actively. The 14 C 3 H ratio was determined in polyribosomes as a measure of the relative ability of the two kinds of ribosomes to initiate protein synthesis. The 14 C 3 H ratio in polyribosomes was found to be identical to the input of labeled ribosomes, indicating an equal ability of ribosomes from growing cells and glutamine-deprived cells to function in initiation. Control studies indicated lack of significant dephosphorylation of S6 during purification of ribosomes and during incubation in the reticulocyte lysate. Thus, a functional difference between more and less phosphorylated ribosomes could not be demonstrated in this system.

Arrangement of the subunits in the ribosome of Escherichia coli: demonstration by immunoelectron microscopy.

The three-dimensional locations of Escherichia coli ribosomal proteins S13, L1, and L7/L12 on the surface of ribosomal subunits and 70S monomeric ribosomes were determined by electron microscopy of antibody-labeled ribosomal particles. A new approach to orient the subunits within 70S ribosomes was developed that used 30S.70S.50S triples that were prepared by simultaneous combination with one antibody directed against a 30S protein and another directed against a 50S protein. Electron microscope studies of triples obtained with the antibody combinations anti-S13/anti-L1 and anti-S13/anti-L7/L12 showed that, in 70S monomeric ribosomes, the head of the 30S subunit is proximate to protein L1 and the peptidyl transferase center but far from the rod-like appendage containing proteins L7 and L12.

Surface features and handedness of a model for the eukaryotic small ribosomal subunit

A three-dimensional model with the appropriate handedness for the small (40 S) eukaryotic ribosomal subunit has been derived from electron microscopic observations of: (a) profiles of negatively stained particles and changes caused by tilting experiments, and (b) surface features of the subunits revealed by metal shadowing. The 40 S subunit is an elongated (270 × 180 × 160A) asymmetric and slightly bent particle which consists of two main regions separated by a circumaxial groove. The upper one-third is oval-shaped and the lower two-thirds has a wedge-shaped bottom portion. A prominence projects upward from the central portion of the subunit. A direct comparison between the features of eukaryotic rat liver 40 S and prokaryotic Escherichia coli 30 S subunits shows similarities in the central and upper one-third portions since main differences in overall size and shape seem primarily confined to the bottom portions of the subunits. In our previous work ( I. Emanuilov, D. Sabatini, J. A. Lake, and C. Freienstein, 1978 , Proc. Nat. Acad. Sci. USA 75 , 1389–1393) the site of attachment of the eukaryotic initiation factor 3 (eIF-3) to the native rabbit reticulocyte 40 S ribosomal subunit was localized on the back side of the central portion of these particles covering part of the large prominence. The same region of the 40 S ribosomal subunit appears to face the 60 S subunit in active monomeric ribosomes and thus is likely to play a functional role in the initiation of protein synthesis.

Bound Ribosomes of Pea Chloroplast Thylakoid Membranes: Location and Release in Vitro by High Salt, Puromycin, and RNase

The mode of attachment of 70S ribosomes to thylakoid membranes from pea leaves was studied by determining the proportion of the bound RNA which was released by various incubation conditions. The results supported a model in which several classes of bound ribosomes could be distinguished: (a) very tightly bound, not released by any conditions yet tested (20% of the total); (b) monomeric ribosomes attached by electrostatic interaction with the membranes (30 to 40% of the total) and released by high salt; and (c) polysomes, with some of the ribosomes attached by a combination of electrostatic interactions and insertion of the nascent polypeptide chain into the membrane. These required a combination of puromycin and high salt for release. Other ("hanging") ribosomes of the polysomes were inferred to be attached through mRNA but not actually attached to the membranes directly; they could be released by RNase under low salt conditions, as well as by puromycin plus high salt.To obtain these results, chloroplasts had to be prepared in media containing 0.2 molar Tris at pH 8.5. Using Tricine buffers at pH 7.5 yielded thylakoid membranes whose ribosomes were removed almost completely by high salt alone; these showed no response to puromycin. However, pH 7.5 had to be used in all cases for ribosome dissociation in high salt media, as the ribosome structure appeared to be degraded by high salt at pH 8.5, and release then occurred without the need for puromycin.The kinetics of ribosome release by high salt showed a rapid initial phase with a half-life of 20 seconds. The extent of release by high salt was very dependent on the temperature of the incubation. Plotting the data according to the Arrhenius interpretation shows a significant break at about 15 C, with apparent activation energy of 20 kilocalories per mole below that temperature and 5 kilocalories per mole above that temperature. This result suggests that membrane fluidity might be an important factor permitting release of ribosomes under high salt conditions.Electron microscope pictures of the washed thylakoids showed polysomes closely associated with the outer membranes of grana stacks, and with the stroma lamellae. Following digitonin treatment of the membranes and centrifugation, fractions enriched in Photosystem I and presumed stroma lamellae were also enriched in bound RNA.

Preparation and analysis of polyribosomes isolated from Tetrahymena pyriformis

1. 1. The Mg 2+ concentration of the solutions used for harvesting cells and preparing postmitochondrial supernatants must be above 4 mM to avoid polysome disaggregation. 2. 2. Sequential reduction of the concentration below this threshold value resulted in a progressive lowering of the polysome size with a concomitant increase in monomeric ribosomes or subunit levels. 3. 3. Changes in KCl concentration or pH value of the isolation buffers did not affect the polysomes. 4. 4. The cell harvesting method used will affect the profiles obtained. For consistent results, the cells must be rapidly chilled prior to harvesting. 5. 5. A variety of cell breakage methods were examined and it was found that some methods produced more reliable results than others. The poorer methods produced smaller classes of polysome suggesting that damage had been sustained by the protein synthetic machinery during isolation. 6. 6. Extracts from different strains of Tetrahymena were examined and the polysome profiles obtained correlated with the cell doubling time. Similar experiments were performed with strain HSM, where the effects of growth medium composition on polysome profiles and on cell doubling times were compared.

The effect of 5-fluorocytosine on the synthesis of 80S ribosomes by pathogenic fungi.

We studied the influence of 5-fluorocytosine(5FC) on the monomeric 80S ribosomes isolated by gradient ultracentrifugation from Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus and Wangiella dermatitidis. Labelling of the cultures with 32PO4 and [3H]-leucine showed marked inhibition of both ribosomal RNA and ribosomal protein synthesized during the contact of the fungi with the drug. The degree of inhibition was very similar for the synthesis of both ribosomal constituents, (mostly 60-80% each during the initial hours of the contact). This indicates that some ribosomes were still synthesized in the presence of 5FC and that the drug inhibits the synthesis of complete ribosomes to the same extent as the synthesis of ribosomal RNA and protein. By labelling fungi with [14C]-5FC it was shown that in those 80S ribosomes that were still synthesized, large quantities of the drug were incorporated--doubtless as 5-fluorouracil (5FU) in RNA. It can be concluded that RNA containing 5FU is still capable of combining with protein to form complete ribosomes of normal conformation. This is, however, no proof that these ribosomes would have a normal function.

Juvenile hormone-controlled vitellogenin synthesis in the fat body of the locust ( Locusta migratoria): Isolation and characterization of vitellogenin polysomes and their induction in vivo

With the aid of EGTA to control the endogenous RNase activity, a predominant population of heavy polysomes was isolated from the fat body of reproductively active female locusts. The identity of these as vitellogenin (Vg)-synthesizing polysomes was established by precipitation of the associated nascent polypeptides with anti-vitellin serum and by translation of the polysomal RNA in Xenopus laevis oöcytes. By EM observation these polysomes were found to contain 40–50 ribosome monomers. In normal development, accumulation of ribosomes in the fat body of female locusts began at about day 6 after adult ecdysis (terminal oöcyte = 2 mm or smaller), and was followed by the appearance of Vg-polysomes beginning at 3 mm terminal oöcyte length (about day 8–10 after adult ecdysis). The content of Vg-polysomes reached a maximum at 5–6 mm oöcyte length (about day 14), and then fell to an undetectable level as the oöcytes reached their maximum size (7 mm). In allatectomized females, Vg-polysomes were induced by treatment with an active juvenile hormone analogue, ZR-515. A single application with ZR-515 produced a massive accumulation of ribosomes and light polysomes in the first 48 hr which was followed by the rapid formation of Vg-polysomes to give a maximum at 72 hr. After the decay of this effect a second dose of ZR-515 resulted in the rapid appearance of Vg-polysomes without the initial generation of ribosomes.

Ribosomal proteins of the cricket, Acheta domesticus L.

The ribosomal proteins of the house cricket, Acheta domesticus, have been characterized using one and two dimensional polyacrylamide gel electrophoresis. Ribosomes isolated from the adult male accessory gland and pelleted through a sucrose cushion contain 85 proteins. Ribosomal monomers from the same source isolated using density gradient centrifugation contain 76 proteins, while large and small subunits contain 38 and 30 proteins, respectively. The molecular weight of proteins associated with ribosomal monomers ranged from 6200 to 77,000. Large subunit proteins had molecular weights ranging from 7600 to 77,000 while small subunit proteins ranged from 8200 to 42,000. Zonal sedimentation of ribosomal monomers through buffer containing 0.5 M KCl resulted in preparations containing 65 proteins. The use of the protease inhibitor, phenylmethylsulfonyl fluoride had no effect on the number or distribution of proteins in gradient purified monomers. A notable feature of undissociated ribosomal monomers is the retention of a number of very low molecular weight proteins of unknown significance; most of these proteins do not appear to be degradation products.

Changes in brain protein synthesis during the life span of male Fischer rats.

The cell-free protein synthetic activity of the postmitochondrial supernatant isolated from whole brain of 6- to 32-month-old male Fischer F344 rats was compared. Protein synthesis decreased 56% from 6 to 32 months of age. The decrease in cell-free protein synthesis was not due to an age-related increase in RNase activity. Although monomeric ribosomes (ribosomes stripped of mRNA) isolated from the brains of older rats were less active in polyuridylic acid directed polyphenylalanine synthesis, the fidelity of polyuridylic acid translation by monomeric ribosomes did not decrease with increasing age.

Discriminant analysis of ribosomal protein synthesis findings in carrier detection of Duchenne muscular dystrophy.

Previous studies have shown that in vitro muscle ribosomal protein synthesis (RPS) by monomeric ribosomes (MR) and total polyribosomes (TPR) and collagen synthesis (CS) are significantly increased (P less than 0.01) in 47 known carriers of Duchenne muscular dystrophy as compared to 60 age-matched controls. However, there was considerable overlap of the distribution of controls and carriers, particularly for monomeric ribosomal protein synthesis and collagen synthesis. To improve detection of carriers we used discriminant analysis utilizing logs of each measurement as superior to a univariate or bivariate scheme. This study considered four groups: proven carriers (30) (group 1), presumptive carriers (female relatives of Duchenne patients with high serum creatine kinase (CK) levels) (32) (group 2), controls greater than or equal to 20 years old (42) (group 3) and controls less than 20 years (36) (group 4). Comparison of groups, Misclassification (%), (see chart). These results suggest that discriminant analysis reduces the misclassification rates as compared with univariate or bivariate analysis and confirm the superiority of RPS measurements as a carrier test for Duchenne muscular dystrophy.

Inactive mRNA-protein complexes from mouse sarcoma-180 ascites cells.

Mouse sarcoma ascites cells do not utilize fully their capacity for protein synthesis. A considerable portion of their ribosomes occur as inactive monomers. Also, a substantial amount of the cellular mRNA is in the form of ribonucleoprotein particles that sediment in the 20-70S range. This is indicated both by measurements of poly(A) content and by translation of the RNA in cell-free systems. The population of polypeptides synthesized under the direction of the RNA from these particles is less heterogeneous than that directed by RNA from polysomes. The mRNAs for some polypeptides are present predominantly in the small particles. Others are distributed to varying degrees between particles and polysomes. Incubation of the cells with cycloheximide drives most of the ribosomal monomers and a portion of the untranslated mRNA into polysomes. Some of the mRNAs that were predominantly in the inactive fraction seem to be refractory to this treatment. Particles released from polysomes in cells subjected to starvation are quite effective in promoting polypeptide synthesis in a reticulocyte cell-free system and cause the synthesis of a population of polypeptides similar to that coded by the polysomal RNA. The particles from cells exposed to cycloheximide are inactive but yield active RNA upon deproteinization. It is suggested that some mRNA species are maintained in an inactive state in the cell by a component of the nucleoprotein complex.

Reassociation of eukaryotic ribosomal subunits by a factor from rat ascites hepatoma cytosol.

Post-ribosomal supernatant extracts from Yoshida AH 130 ascites hepatoma cells promote the in vitro association of ribosomal subunits at low Mg2+ concentration. Comparable extracts from rat liver show, on the contrary, dissociation factor activity on ribosome monomers.

Biosynthesis of ribosomal proteins by poly(A)-containing mRNAs from rat liver in a wheat germ cell-free system and sizes of mRNAs coding ribosomal proteins

(1) Poly(A)-containing mRNAs from total polysomal RNA of regenerating rat liver were incubated with [ 3H]leucine in a wheat germ cell-free system. Ribosomal proteins were purified as described previously [1], and with two-dimensional gel electrophoresis. The proteins on the gel except for less basic protein had appreciable radioactivity, whereas the surrounding areas had very low radioactivity. Acetic acid-soluble proteins labeled in this system were subjected to three-dimensional gel electrophoresis [2]. Except for L1 and L2 proteins, each of the ribosomal proteins, including less basic ones, showed a major radioactive peak coinciding with the protein band on SDS gel. Thus, the wheat germ cell-free system completely translates almost all mRNAs for individual ribosomal proteins. Equimolar amounts of almost all ribosomal proteins were synthesized in the presence of the saturating concentration of mRNAs. (2) Free polysomes from regenerating rat liver were fractionated into three sizes. Each class of polysomes was incubated with [ 3H]leucine. Ribosomal proteins with molecular weights of 40 000 to 21 000 were mainly synthesized by Fraction B (5–14 monomeric ribosomes), L1 and L2 [2] with 60 000 and 54 000, by Fraction C (>15 monomeric ribosomes) and B, and ribosomal proteins smaller than 20 000 by Fractions A (<pentamer) and B. (3) mRNAs from rat liver total polysomes were fractionated into seven classes by size and each was translated in the wheat germ extract. Ribosomal proteins with molecular weights of 54 000 to 30 000 were mainly synthesized by mRNAs of 12 to 14.5 S, ribosomal proteins of 35 000 to 22 000 by those of 9.5 to 12 S, ribosomal proteins of 22 000 to 13 000 by those of 7 to 9.5 S, and smaller ribosomal proteins by those smaller than 7 S. These results indicate that individual ribosomal proteins are synthesized by monocistronic mRNAs, the lengths of which are proportional to the molecular weights of the corresponding ribosomal proteins.

Alteration in thermal stability of ribosomes from Drosophila melanogaster with age.

Thermal analysis of high salt (0.5 M) washed ribosomal monomers from young and old male Drosophila melanogaster revealed an 8 degrees C downshift in the mean temperature of denaturation (Tm). Moreover, there was observed a marked loss in the ability of ribosomes extracted from older flies to reassociate upon cooling. These observations suggest that age-dependent alterations in the structural integrity of the rRNA-r protein complex could, at least in part, be responsible for the diminished capacity for protein synthesis in this species with advancing age.

The association of histidyl-tRNA synthetase with reticulocyte ribosomes

Some aminoacyl-tRNA synthetase activity is found attached to ribosomes. This phenomenon was investigated in detail using the histidyl-tRNA synthetase of rabbit reticulocytes, 10–15% of which is attached to ribosomes from cells lysed osmotically. The enzyme can be eluted from ribosomes by KCl solutions of 0.1 to 0.2 M. The enzyme eluted from ribosomes and the enzyme of reticulocyte cytosol can be associated with KCl washed ribosomes. A constant ( K A ) of 5 · 10 8 to 5 · 10 9 M −1 has been determined for the association reaction, indicating a high level of affinity and specificity. One bound enzyme molecule is found per 500 ribosomes from freshly lysed reticulocytes, and one molecule can be associated per 300 ribosomes from a crude enzyme preparation. One enzyme molecule can be associated per two ribosomes using a partially purified enzyme preparation. Thus the number of binding sites approaches a limit, and possibly molecules in addition to His-tRNA synthetase can be attached to the same binding sites. Sucrose density gradient centrifugation indicates that the enzyme is bound both to polysomes and to 80-S ribosome monomers, and that the enzyme is bound by each of the ribosomal subunits. There appear, therefore, to be at least two binding sites per ribosome. The significance of ribosomal binding of aminoacyl-tRNA synthetases, which occurs with high affinity and specificity in the absence of KCl, is considered.

Mutations in nine chloroplast loci of Chlamydomonas affecting different photosynthetic functions.

Chloroplast components known to be coded by chloroplast DNA include chloroplast rRNAs, tRNAs, and the large subunit of ribulose-bisphosphate carboxylase. Because these components comprise less than 3% of the estimated coding capacity of the chloroplast genome, most chloroplast gene functions have yet to be identified. One approach to this problem is the isolation and characterization of mutations in the chloroplast genome affecting specific photosynthetic functions. Recently we have found that such mutations can be preferentially recovered by using arsenate selection on cells previously grown in 5-fluorodeoxyuridine. Sixteen mutants thus isolated have been localized into nine chloroplast loci, based on their ability to recombine and produce photosynthetically competent progeny. Mutants at two loci show the characteristic syndrome of photosynthetic defects that results from a deficiency in chloroplast protein synthesis. These have been found to lack chloroplast ribosome monomers. Mutants at three loci are missing chlorophyll-protein complex I in their thylakoid membranes. Mutants at three other loci are deficient in membrane polypeptides known to be associated with the chloroplast coupling factor.

Activity of Thylakoid-bound Ribosomes in Pea Chloroplasts

Pea (Pisum sativum) chloroplast thylakoid membranes were prepared by washing in hypotonic buffers. These membranes contained bound ribosomes which were active in protein synthesis when supplemented with soluble components from a strain of Escherichia coli low in ribonuclease. After dissolving the membranes by Triton and purification of the ribosomes, sucrose density gradient profiles indicated the presence of polysomal material as well as monomeric ribosomes. Most of the products of protein synthesis remained associated with the thylakoid membranes even after ribosomes were removed completely by high salt concentrations in the absence of Mg(2+). Of the newly formed products, 50% could be digested by pronase, while the remainder were protected by their association with the thylakoid membranes. The products are likely to be a mixture of intrinsic and extrinsic membrane proteins, with only the former completely protected by the membranes from attack by proteases.

Alteration of hepatic polyribosome structure and function in mice during hypothermia

Analysis of the distribution of RNA double labeled with [ 14C]- and [ 3H]orotic acid in ribosomal particles separated by sucrose density-gradient centrifugation and the selective dissociation of unprogrammed ribosomes showed that the increase in proportion of monomeric ribosomes caused by hypothermia is due entirely to the increase in the unprogrammed ribosome fraction at the expense of polyribosomes. Protein synthetic activities of polyribosomes in vivo in livers of hypothermic and control mice were compared by double labeling of nascent and soluble polypeptides with [ 14C]- and [ 3H]leucine. Incorporation of radioactivity was relatively higher in nascent polypeptides and lower in soluble polypeptides in hypothermic than in control mice. The results indicate that hypothermia affects protein synthesis in livers of mice by decreasing probably the rates of both initiation and elongation, but affecting the former more than the latter.

In vivo effect of colchicine on hepatic protein synthesis and on the conversion of proalbumin to serum albumin.

Treatment of rats with 0.5-25 mumol/100 g body weight of colchicine for 1 h or more caused an inhibition of hepatic protein synthesis. This effect was not seen if animals were exposed to colchicine for less than 1 h. The delayed inhibition of protein synthesis affected both secretory and nonsecretory proteins. Treatment with colchicine (15 mumol/100 g) for 1 h or more caused the RNA content of membrane-bound polysomes to fall but did not change the polysomal profile of this fraction. By contrast, the total RNA content in the free polysome cell fraction was increased, and this was due to the presence of more ribosomal monomers and dimers. Electron microscope examination of the livers from rats treated for 3 h with colchicine showed an accumulation of secretory vesicles within the hepatocytes and a general distention of the endoplasmic reticulum. Administration of radioactive L-leucine to the rats led to an incorporation of radioactivity into two forms of intracellular albumin which were precipitable with antiserum to rat serum albumin but which were separable by diethylaminoethyl-cellulose chromatography. One form has arginine at the amino-terminal position and is proalbumin, and the other form, which more closely resembles serum albumin chromatographically, has glutamic acid at its amino terminus. Only proalbumin was found in rough and smooth endoplasmic reticulum fractions and in a Golgi cell fraction wich corresponds morphologically to mostly empty and partially filled secretory vesicles. However, in other Golgi cell fractions which were filled with secretory products, both radioactive proalbumin and serum albumin were found. This indicates that proalbumin is converted to serum albumin in these secretory vesicles just before exocytosis. Colchicine delayed the discharge of radioactive albumin from these filled secretory vesicles and caused an accumulation of both proalbumin and serum albumin within these cell fractions.

Mutual Relations between Typical Images of Negatively Stained Ribosomal Monomer and Large Subunit Particles from &lt;italic&gt;E. coli&lt;/italic&gt;: An Electron Microscopic Study with Tilting Technique

Mutual Relations between Typical Images of Negatively Stained Ribosomal Monomer and Large Subunit Particles from &lt;italic&gt;E. coli&lt;/italic&gt;: An Electron Microscopic Study with Tilting Technique