Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells. A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results. This procedure is readily adaptable to mammalian cells.     

Ribosomal complexes from an extremely halophilic bacterium and the role of cations

Concentrated extracts of Halobacterium cutirubrum were prepared at 0 C by gently disrupting cells with a nonionic detergent in a medium containing 3.0 m KCl, 0.5 m NH(4)Cl, and 0.04 m (or more) magnesium acetate and then treating the gelatinous mass with deoxyribonuclease. On KCl-sucrose gradients containing 0.5 m NH(4)Cl and 0.04 m magnesium acetate, these extracts showed 30S and 50S ribosomal subunits plus a flat profile of faster-sedimenting material up to high S values. Only after frozen storage or brief incubation of the extract were 70S ribosomes and distinct classes of small polyribosomes detected. Digestion with ribonuclease converted faster-sedimenting material to 70S particles. The presence of chloramphenicol during preparation of the extracts did not affect these results. The evidence suggests that ribosomal particles exist in these cells as subunits or as polyribosomes but not as 70S ribosomes. To investigate the function of Mg(++) and NH(4) (+) ions in ribosomal complexes from this halophile, concentrated cell extracts and extracts incubated with (14)C-leucine were examined on KCl-sucrose gradients containing different concentrations of these ions. Polyribosomes and the bulk of 70S ribosomes dissociated reversibly to subunits at about 0.01 m Mg(++), whereas a small fraction of the 70S particles, including those which in vitro incorporated (14)C-leucine into nascent protein, dissociated only below 1 mm Mg(++). Below this concentration of Mg(++), nascent protein remained attached to the 50S subunit even at 0.04 mm Mg(++) in the presence of 0.35 to 0.5 m NH(4)Cl. Nascent protein, presumably as peptidyl-transfer ribonucleic acid, dissociated reversibly from 50S subunits only at 0.04 mm Mg(++) and 0.1 m or less NH(4) (+). Thus, the stability of polyribosomes from H. cutirubrum depends specifically on both Mg(++) and NH(4) (+) ions.     

A STRUCTURAL STUDY OF RAT LIVER RIBOSOMES

Polyribosomes, ribosomes, and ribosomal subunits were prepared from rat liver using sodium deoxycholate and a variety of ionic media. They were examined in the electron microscope, mainly as negatively or positively stained preparations, and in the analytical ultracentrifuge. The polyribosomes consist of up to twelve or more ribosomes linked by a fine strand, 10 to 15 A in diameter, probably of RNA. The ribosomes are approximately spherical with diameters of 250 to 300 A, and are estimated to be about 50 per cent porous. Possibly because of their high protein content, whole ribosomes show no cleavage furrows. Ribosomes were dissociated in phosphate buffer and the subunits separated on sucrose density gradients containing 10 per cent formalin. Three classes of subunit were obtained with sedimentation coefficients of 71S, 50S, and 31S respectively. The smallest, 31S subunit is about 250 A long by 100 A wide. The largest subunits appear to be clusters of smaller particles. It is estimated from their linear dimensions in electron micrographs that the whole 83S ribosome could contain up to six 31S subunits, or their equivalent.     

The mechanism of polyribosome disaggregation in brain tissue by phenylalanine.

The injection of neonatal mice with phenylalanine resulted in a rapid decrease in brain polyribosomes and a concomitant increase in monomeric ribosomes. Animals of 1-16 days of age were equally affected by phenylalanine, although the brain polyribosomes of 60-day-old mice were relatively resistant to the effects of phenylalanine. The population of free polyribosomes appeared to be more sensitive to phenylalanine treatment than bound polyribosomes, which were somewhat more resistant to disruption by high concentrations of the amino acid. The effects of phenylalanine were more pronounced with polyribosomes in the cerebral cortex than with those in the cerebellar tissue. The mechanism of polyribosome disruption was shown to be independent of hydrolysis mediated by ribonuclease. Virtually all of the monomeric ribosomes that resulted from phenylalanine treatment were shown to be inactive with regard to endogenous protein synthesis and were present in the cell cytoplasm as vacant couples. These ribosomes were readily dissociated by treatment with 0.5 M-KCl and subsequent ultracentrifugation. These results are discussed in the light of the possibility that high concentrations of phenylalanine disrupt brain protein synthesis by a molecular mechanism that is associated with initiation events.     

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats.

1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [114C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell cap. 6. The results are discussed in relation to the problem of the control of protein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.     

Ribosome function in livers of porphyric mice.

1. A porphyrinogenic drug, 3,5-diethoxycarbonyl-1,4-dihydrocollidine, caused a decrease in the proportion of single ("run off") ribosomes, and an increase in the number of polyribosomes, in the livers of treated animals. 2. No change could be detected in the distribution of amino acid incorporation among hepatic polyribosomes.     

THE IN VIVO PROTEIN SYNTHETIC ACTIVITIES OF FREE VERSUS MEMBRANE-BOUND RIBONUCLEOPROTEIN IN A PLASMA-CELL TUMOR OF THE MOUSE

Cytoplasmic extracts of the transplantable RPC-20 plasma-cell tumor were fractionated by sucrose density gradient centrifugation. Four major fractions were distinguished: (a) microsomes and mitochondria; (b) membrane-free polyribosomes; (c) free monomeric ribosomes; and (d) soluble fraction. The fractions were analyzed for RNA and lipid phosphorus, and their particulate components were characterized by electron microscopy. Particular attention was paid to the problem of membrane contamination of the free polyribosome fraction. It was shown that this contamination was small in relation with the total content of ribosomes in the fraction, and that it consisted primarily of smooth-surfaced membranes which were not physically associated with the polyribosomes themselves. In vivo incorporation studies were carried out by injecting tumor-bearing animals intravenously with leucine-C¹⁴, removing the tumors at various times thereafter, and determining the distribution of protein radioactivity among the gradient-separated cytoplasmic fractions. The free polyribosome and the microsome-mitochondria fractions constituted active centers for protein synthesis. It was shown that nascent protein of the free polyribosome fractions was not associated significantly with the contaminating membranes. The kinetics of labeling during incorporation times up to 11 min suggested that protein synthesized on the free polyribosomes was rapidly transferred in vivo to the soluble fraction of the cell, while protein synthesized by the microsomes and mitochondria remained localized within these elements. It was estimated that the free polyribosome fraction and the microsome-mitochondria fraction accounted for approximately equal proportions of the total cytoplasmic protein synthesis in vivo.     

Amino acid incorporation by ribosomes and polyribosomes from wheat chloroplasts

Sucrose-gradient and analytical ultracentrifugation showed that chloroplast polyribosomes from 4-day-old seedlings had mono-, di-, tri-, tetra- and traces of penta-ribosomes, in contrast with those from 7-day-old seedlings in which only the mono-, di- and traces of tri-ribosomes were present. Without Mg(2+) the polyribosomes dissociated into ribosomal subunits. The rate of l-[U-(14)C]phenylalanine incorporation was threefold greater for preparations from 4- than from 7-day-old seedlings. Incorporation by the latter was stimulated by polyuridylic acid. The rates of incorporation were similar whether the reaction mixture contained chloroplast or wheat-germ transfer RNA and amino acid synthetases purified on methylated albumin-on-kieselguhr and Sephadex G-75 columns respectively. The cofactor requirement was the same as for isolated intact chloroplasts. Osmotic rupture of chloroplasts with and without Triton X-100 revealed the presence of free and bound ribosomes. Free single ribosomes isolated by osmotic shrinkage or prepared by pancreatic ribonuclease digestion of chloroplast polyribosomes had negligible incorporation activity. This activity was increased by washing or by polyuridylic acid, but was still only a fraction of that given by polyribosomes. A comparison of incorporation activity of chloroplast polyribosomes with those from the surrounding cytoplasm showed the former to be 20 times more active.     

The synthesis of α-amylase by rough and in vitro reconstituted rough endoplasmic reticulum derived from rat parotid gland

Summary The isolation of rough and smooth endoplasmic reticulum from rat parotid salivary gland is described. The rough membrane was stripped of its bound ribosomes using the KCl-puromycin method. Rough endoplasmic reticulum was reconstituted from stripped-rough membrane and polyribosomes. The reconstituted rough membrane resembled the native rough membrane in the following aspects: RNA/protein ratio, buoyant density in a continuous sucrose gradient and amino acid incorporation capacity. The in vitro synthesis of -amylase by both rough and in vitro reconstituted rough membrane was demonstrated using SDS polyacrylamide gel electrophoresis. The reconstituted rough membrane could be restripped by KCl-puromycin. The in vitro synthesized -amylase remained associated with the rough or the in vitro reconstituted rough membrane, even after these membranes were stripped of their bound ribosomes.Abbreviations Fp Free polyribosomes - Bp Membrane-bound polyribosomes released by DOC - RM Rough membrane - SM Smooth membrane - RMst Rough membrane stripped - RMrec In vitro reconstituted rough membrane - DOC Sodium deoxycholate     

The in vivo effect of dimethyl sulphoxide (DMSO) on protein synthesis and the polyribosome profile in Paramecium.

When Paramecium tetraurelia in log phase growth is treated with 4% dimethyl sulphoxide (DMSO) for five minutes the amount of polyribosomes is reduced 3- to 4-fold while there is a corresponding increase in 80s ribosomal material. Reducing the concentration of DMSO to 1% allows immediate reversal of the condition. Paramecium polyribosomes subjected to 4% DMSO either in whole cell homogenates or during purification through sucrose density gradients appear unaffected while cycloheximide at concentrations up to 100 mug/ml did not prevent DMSO from exerting its effect in vivo. Analyses of 14C amino acid incorporation experiments indicated a strict correspondence between the effect of DMSO on polyribosomes and overall protein synthesis. The reduction of acid precipitable radioactivity in the polyribosomal region after DMSO treatment was associated with a corresponding increase in radioactivity in the 80s region. There was no comparable increase in the acid precipitable radioactivity in the soluble fraction. The overall results of the study suggest that DMSO acts on polyribosommes indirectly through some unknown primary reaction with cell constitutents, and that the mode of action is such as to cause the release of ribosomes from messenger RNA (mRNA) rather than to prevent initiation of the ribosome-mRNA complex. Our data suggest that the effect may be selective. Finally, it is of interest that high concentrations of DMSO (above 8%) appear to have the opposite effect of lower concentrations of DMSO, i.e., they appear to "freeze" the ribosomes to mRNA.     

Association of D-RNA with Polyribosomes in the Soybean Root

Ribosomes from soybean root tips were shown to consist of about 75% polyribosomes based on size distribution on sucrose gradients. Electronmicrographs showed the presence of ribosome clusters in normal preparations and only monomers and dimers in ribonuclease-treated preparations. The polyribosome preparations, but not single ribosomes, showed good in vitro amino acid incorporating activity.     

Presence of active polyribosomes in bacterial cells infected with T4 bacteriophage ghosts.

Host protein synthesis of Escherichia coli stops abruptly after T4 bacteriophage ghost infection. When infection was carried out in the presence of 10 mM Mg2plus, infected cells still have active polyribosomes despite the complete stoppage of protein synthesis. On the other hand, when T4 ghost infection was carried out in the presence of 1 mM Mg2plus, no polyribosomes were observed and most of the ribosomes were 30S and 50S subunit particles. Subunits obtained from extracts of ghost-infected cells at 1 mM M'G2++ concentration could not be converted to polyribosomes, even when Mg2plus concentration was adjusted to 10 mM after ghost infection. There was very little difference in amino acid incorporation activities between polyribosomes from ghost-infected and uninfected cells. In addition, the activity of 70S ribosomes isolated from uninfected cells was identical to that from cells infected with ghosts at 10 mM Mg2plus.     

Factor-dependent binding of Rauscher leukemia virus RNA to ribosomes

Rauscher leukemia virus (RLV)-³H-RNA was shown to bind to ribosomes with the sedimentation properties of polyribosomes when incubated in a cell-free amino acid incorporation system derived from RLV-infected JLS-V5 cells. The binding of RLV-³H-RNA to ribosomes requires factors present in the high-salt wash of JLS-V5 polyribosomes, and is inhibited by aurintricarboxylic acid (ATA). The RLV-³H-RNA-ribosome complexes are sensitive to ribonucleases (RNase), ethylenediaminetetraacetate (EDTA), and puromycin.     

Protein Synthesis by Free and Bound Paramecium Ribosomes in Vivo and in Vitro

SYNOPSIS. Cytoplasmic extracts of Paramecium aurelia were fractionated by density gradient centrifugation. The gradient fractions were characterized by chemical analysis and electron microscopy. Membrane-bound ribosomes were separated from free polyribosomes and the ability of each of these forms to incorporate C¹⁴-leucine into protein was tested. Incorporation was measured in both in vivo and in vitro systems, and similar results were obtained in both types of experiment except that there was little release of soluble labelled protein in the in vitro system. Paramecium appears to synthesize most of its protein on free polyribosomes but membrane-bound ribosomes constitute an important protein synthetic fraction, perhaps accounting for as much as 30% of the total synthesis. When isolated in the in vitro system, increasing concentration of the ribosome fraction gave increased incorporation, but increasing concentration of the membrane fraction gave decreased incorporation after a critical value. This inhibitory effect can be removed by adding excess cytoplasmic-supernatant to the system. The nature of the association of ribosomes with membranes is discussed.     

Polyribosomes in rat-liver preparations

1. The distribution of rat-liver polyribosomes in sucrose density gradients has been investigated with regard to the effects of the preparative procedures and the physiological and pathological condition of the animal. 2. By using carefully defined conditions, three principal polyribosomal fractions have been isolated with S(20,w) values of 340, 275 and 225s in addition to the dimerized 120s and single 80s ribosomes. 3. The polyribosomes were very sensitive to treatment with ribonuclease and to mechanical stresses. 4. Incubation of dispersed hepatic cells and also cell-free preparations with puromycin in the presence of ATP and phosphoenolpyruvate caused rapid partial degradation of the polyribosomes. Treatment of the dispersed cells with actinomycin D also degraded the polyribosomes. 5. The liver polyribosomes of rats not raised under pathogen-free conditions and possibly of rats with an arthritic syndrome may be more fragile than those of healthy pathogen-free animals. 6. Treatment of pathogen-free rats with drugs stimulating liver anabolism profoundly affected the distribution of polyribosomes in sucrose density gradients.     

Functional modification of rat liver ribosomes by the in vitro action of N-methyl-N'-nitro-N-nitrosoguanidine

The mechanism by which N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) inhibits protein synthesis has been studied in a rat liver cell free system. Using preformed aminoacyl-tRNA it was observed that incorporation of amino acid into polyribosomal protein was inhibited in the presence of low concentration of MNNG. This inhibition was not reversed by increasing the concentration of soluble factors. Transfer RNAs modified previously by treatment with MNNG and subsequently esterified with amino acids were transferred to polyribosomes with the same efficiency as those species which were not modified. Polyribosomes, on the other hand, lost activity to incorporate amino acids after pretreatment with MNNG. This inactivation was dependent on the concentration of MNNG with which polyribosomes were treated. When poly(U) was used with MNNG-treated polyribosomes, its translation, after correction for endogenous translation, was also found to be significantly low as compared to the case with untreated polyribosomes. Purified ribosomes stripped of endogenous mRNA when treated with increasing concentrations of MNNG progressively lost ability to support polyphenylalanine synthesis programmed by poly(U). The treated ribosomes, however, neither inhibited the activity of control ribosomes nor induced any loss of fidelity of translation by poly(U). It is concluded that MNNG inhibits protein synthesis through functional inactivation of ribosomes resulting from direct modification of ribosomal proteins possibly involving nitroguanidination of lysine residues.     

Response of Cultured Mammalian Cells to Diphtheria Toxin III. Inhibition of Protein Synthesis Studied at the Subcellular Level

Diphtheria toxin inhibited protein synthesis in intact KB cells. The action of the toxin upon the cell did not result in disaggregation of polyribosomes, or in impairment of their ability to function in protein synthesis. A reduction in single ribosomes and a concomitant increase in polyribosomes did result from the action of toxin. Nascent peptides were not cleaved from polyribosomes by the action of toxin, but treatment of fully intoxicated cells with puromycin resulted in cleavage of these peptides, and caused accelerated polyribosome breakdown. Our data indicated that the toxin must enter the cell to exert its effect. The component or components sensitive to toxin were localized in the 100,000 x g supernatant fraction of cytoplasmic extracts. When extracts from intoxicated cells were treated with nicotinamide, a significant proportion of their capacity to synthesize protein was restored. The specificity of this reaction suggested that nicotinamide adenine dinucleotide is involved in the action of toxin in the intact cell, and that one component inactivated by toxin is soluble transferase II.     

RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.     

Cytoplasmic effects of cortisol in liver

1. The time-course of the induction by cortisol of tryptophan pyrrolase in mouse liver was established. 2. The kinetics of labelling of mouse liver polyribosomes and the distribution of radioactivity in the various sizes of polyribosomes were investigated. It is concluded that the major part of the messenger RNA is monocistronic. 3. Cortisol administered to adrenalectomized mice stimulates in parallel the ability of ribosomes and polyribosomes, both free and bound, to incorporate amino acids into protein. 4. This stimulation does not appear to affect in a selective manner particular regions of the gradients of polyribosomes. 5. Administration of cortisol enhances the ability of the denser fraction of the rough microsomes to incorporate labelled amino acids. Adrenalectomy has minor effects on the pool sizes and specific radioactivities of liver amino acids which are counteracted by the hormone. However, these appear insufficient to influence the changes observed. 6. Adrenalectomy causes an increase in the percentage of ribosomes existing as polyribosomes and a fall in the proportion bound to the membrane of the endoplasmic reticulum. After treatment of these animals with cortisol there is a marked increase in the proportion of bound ribosomes. 7. The influence of cortisol on the membranous components of liver cytoplasm is discussed.     

The in vitro reconstitution of a functional rough membrane active in protein synthesis

Rough endoplasmic reticulum was reconstituted from free polyribosomes and rough membrane stripped from its ribosomes by KCl and puromycin. The reconstituted rough membrane resembled the native rough membrane in the following aspects: RNA/protein ratio, buoyant density in a continuous sucrose gradient, amino acid incorporation capacity and sensitivity towards protein synthesis inhibitors. When the reconstitution was done with double labelled polyribosomes ([³²P] polyribosomes, [³H] leucine labelling of nascent peptide chain before or after the attachment of the polyribosomes to the membrane) both labels banded together with the reconstituted rough membrane band. Hybrid rough membrane could be formed from rat liver stripped rough membrane and wheat germ ribosomes. This hybrid membrane could translate globin mRNA.