STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES : IV. Some Observations on the Properties of Ribosomes and Polysomes from Rat Liver and Hepatomas

Polysome and ribosome preparations from normal rat liver and from a series of transplantable rat hepatomas of different growth rates were compared. All the hepatomas had a significantly higher percentage of RNA in a polysome preparation than did the normal liver, and the polysome preparations from the tumors, with the exception of the Dunning hepatoma which has a high lipid content, gave a greater yield of RNA and protein per gram of wet tissue than the liver did. Heavier polysomes were considerably less prevalent in the tumors than in the liver, and the tumors contained a larger proportion of monomer and dimer ribosomes than the liver did. Evidence is presented that the increased monomer and dimer ribosome population of the hepatomas studied is not an artifact of preparation, but represents the true intracellular distribution. Ribosomes from normal liver and Morris 5123-D hepatoma were readily dissociated by 20 min'' treatment with 1.0 mM EDTA, but ribosomes from the Dunning, Novikoff ascites, and McCoy MDAB hepatomas were little affected by such treatment. With higher concentrations of EDTA, the ribosomes from the Novikoff ascites and McCoy MDAB hepatomas broke down and did not form specific subunits as did ribosomes from liver and the Morris 5123-D hepatoma but rather gave rise to a variety of small degradation products. This behavior is ascribed to a higher RNase content of the Novikoff and McCoy MDAB hepatomas. Dunning hepatoma ribosomes were resistant to 4 mM EDTA.     

Ribosome bound ribonuclease; its preferential association with small polysomes

Ribonuclease of the total rat liver ribosome fraction proved to be considerably more active than the same enzyme of the polysome fraction. This diminished polysomal activity was caused by exclusion of the enzymerich small polysomes and monosomes from discontinuous sucrose gradient preparations. An incidental finding was the demonstration that regenerating liver ribosomes appear to carry some of this enzyme in a dormant state not normally revealed during autodegradation.     

Comparision of the structure and function of polysomal and helical ribosomes from Entamoeba invadens

Some structural and functional properties of ribosomes from polysomes and from helix aggregates of Entamoeba invadens have been compared by sucrose gradient analysis and assays of in vitro protein synthesis. Actively growing trophozoites, lacking helices, presented normal polysome profiles in sucrose gradients. The single large ribosomal helix aggregate (chromoatoid body) of cysts diappeared as the cells were disrupted. Gradient profiles of cyst extracts contained predominantly large and small ribosome subunit peaks and no evidence of remaining helix fragments of mRNA-bound polysomes. Sequential profiles of trophozoites incubated with NaF or cycloheximide (which both stimulate ribosome aggregation, but at different rates) showed that polysome breakdown occurred before aggregates appeared and, again, that helices broke down to subunits in vitro. Radioactive ribosomes synthesized during vegetative growth were collected into helices during encystation. Subunits of these ribosomes cosedimented with comparable particles isolated from trophozoites. Ribosomes from both trophozoites and cysts were active in cell-free protein synthesis, although activity in cyst extracts required the addition of trophozoite-soluble fraction. It was concluded that ribosomes from polysomes and helices in E. invadens were probably identical and that the ability to form helices was an intrinsic property of mature mRNA-free ribosomes of this organism.     

RNase catalyzed hydrolysis of ribosomes in several functional states

The RNase A catalyzed hydrolysis of rRNA in ribosomes has been studied for nonwashed 50S and 70S ribosomes, for washed 50S and 70S ribosomes, for runoff 50S ribosomes and for 70S ribosomes in polysomes. The regions available to hydrolysis in the 50S ribosome remain available when the 50S ribosomes become a part of a 70S ribosome or a polysome. The regions available to hydrolysis in the 30S ribosome become unavailable when the 30S ribosome becomes part of a 70S ribosome or a polysome. Removal of tRNA, mRNA and factors from the 50S and 70S ribosome lowers the rate of hydrolysis of one site in the 23S rRNA. This shows that the conformation of one region of the 23S RNA changes for ribosomes in different functional states.     

Direct association of messenger RNA-containing ribonucleoprotein particles with membranes of the endoplasmic reticulum in ethionine-treated rat liver.

The administration of ethionine to female rats causes breakdown of hepatic polysomes. The fate of the mRNA molecules after polysome breakdown was investigated by measuring the amount of poly(A)-containing mRNA in membranous and non-membranous fractions obtained from the cytoplasm of ethionine-treated rat liver. The amount of poly(A)-containing mRNA in the membrane fraction of ethionine-treated liver was found to be the same as that of normal liver. When poly(A)-containing mRNAs from various fractions were translated in a wheat germ system and the products were isolated by immunoprecipitation, the albumin-specific mRNA was found exclusively in the membrane fraction of both normal and ethionine-treated livers. The membrane-bound mRNA in ethionine-treated liver, selectively labeled with [14C]orotate, was banded in CsCl gradient centrifugation at 1.42 g/ml which corresponds to the previously reported mRNA-containing ribonucleoprotein particles. From these results, we concluded that even after the polysome disaggregation by ethionine, most of the mRNA of membrane-bound polysomes remains attached to the endoplasmic reticulum membranes independently of ribosomes and the nascent polypeptide chains.     

fMet-tRNA F Met binding and peptidyl transferase function in free and bound ribosomes from normal and puromycin aminonucleoside-treated rats.

Treatment of rats with the aminonucleoside of puromycin, which increases the incorporation of labelled phenylalanyl-tRNA into polypeptide chains in liver ribosome preparations studied in vitro, did not change the factor-dependent binding of fMet-tRNA f Met to ribosomes nor the peptidyl transferase function of the ribosomes. Peptidyl transferase function, as measured by fMet-tRNA f Met-puromycin formation, was comparable in the free and bound ribosome preparations. Similarly, the factor-dependent binding of fMet-tRNA f Met to ribosomes was the same in free ribosome preparations obtained from rat liver as it was in bound ribosome preparations that had been freed of membranes by puromycin incubation and high salt wash.     

STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES : III. The Relationship of the Ribonuclease Activity of Rat Liver Microsomes to their Biological Activity

Attempts have been made to prepare rat liver microsomes and ribosomes free of RNase activity. Washing of microsomes with a large number of reagents, as well as preparation of microsomes by homogenizing the liver in the presence of a variety of reagents chosen to remove or inhibit RNase activity, failed to abolish completely the enzyme activity. However, when rat liver was homogenized in the presence of optimal concentrations of ATP the microsomes subsequently obtained showed no RNase activity. The composition of such microsomes was compared to controls prepared without the use of ATP. Preparation of microsomes with the use of ATP apparently repressed but did not remove the RNase activity for, when such microsomes were treated with 1 per cent deoxycholate to obtain ribosomes, the latter exhibited normal RNase activity. A possible explanation for these results based on several experiments is given. The incorporation of C¹⁴ of L-leucine-C¹⁴ into control and ATP-treated microsomes was measured. Repression of RNase activity by use of ATP or with RNase inhibitor, significantly reduced the incorporation. As a result of these and other experiments it is tentatively concluded that an alkaline RNase is a normal constituent of rat liver ribosomes and plays a role in the biological activity of these particles.     

Influence of amino acid supply on ribosomes and protein synthesis of perfused rat liver

1. The livers of rats were perfused in situ with medium containing mixtures of amino acids in multiples of their concentration in normal rat plasma. The incorporation of labelled amino acid into protein of the liver and of the perfusing medium increased with increasing amino acid concentration. During 60min. perfusions, labelling of liver protein reached a plateau, and labelling of medium protein was inhibited when the initial concentration of the amino acid mixture was more than ten times the normal plasma value. 2. Examination of polysome profiles derived from livers perfused without amino acids in the medium showed that the number of large aggregates was decreased and the number of small aggregates, particularly monomers and dimers, was increased with time of perfusion. The addition of amino acids to the perfusion medium reversed this polysome shift to an extent that was dependent on the initial concentration of amino acids. Polysome profiles derived from livers perfused for 60min. with ten times the normal plasma concentration of amino acids were essentially the same as the polysome profiles of normal non-perfused livers. 3. The ability of ribosome preparations from perfused livers to incorporate amino acids into protein in vitro decreased with increasing time of perfusion when no amino acids were added to the medium, but increased as the concentration of amino acids in the perfusion medium was increased. 4. The ability of cell sap from perfused livers to support protein synthesis in vitro was not influenced by the amino acid concentration of the perfusion medium. 5. Livers were perfused for 60min. with medium containing amino acid mixtures at ten times the normal plasma concentration but deficient in one amino acid. Maximal incorporation of labelled amino acid into liver protein, the stability of the polysome profile and the ability of ribosome preparations to incorporate amino acids into protein were found to depend on the presence of 11 amino acids: arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine. A mixture of these 11 amino acids, at ten times their normal plasma concentration, stimulated the incorporation of labelled amino acid into liver protein, stabilized the polysome profile and increased the ability of ribosome preparations to incorporate amino acids into protein to the same extent as the complete mixture. 6. It is concluded that the availability of certain amino acids plays an important role in the control of protein synthesis, possibly by stimulating the ability of ribosomes to become, and to remain, attached to messenger RNA.     

Initiation of mammalian protein synthesis: the importance of ribosome and initiation factor quality for the efficiency of in vitro systems

A highly efficient mammalian system was developed for the in vitro translation of exogenous rabbit globin messenger RNA. The system consists of purified ribosome subunits from mouse liver, rabbit reticulocytes, or guinea pig brain, partially purified initiation factors from rabbit reticulocytes, and elongation factors, termination factors, aminoacyl-tRNA synthethases and tRNA from rat liver in the form of pH 5-enzymes. (1) Emphasis was put on well-defined, structurally and functionally intact ribosomes, which we found to be the most difficult component of the system in terms of stability of activity. (2) An improved method for extraction of initiation factors from crude reticulocyte ribosomes was developed. Factor preparations of high specific activity were obtained by a simple, partial purification procedure. The crucial point was not to damage the ribosome structure during extraction of the initiation factors and to eliminate inhibitory components during extraction and purification. (3) The efficiency of the system was demonstrated quantitatively by showing that between $\text{3}\text{4}$ and one mRNA molecule per ribosome saturates the system and that each ribosome recycles over the mRNA several times. (4) Major uncertainties and ambiguities in the search for and identification of true initiation factors, as opposed to structural ribosome proteins needed for the reconstitution of damaged ribosomes, can be reduced by using this system.     

Functional Escherichia coli 23S rRNAs containing processed and unprocessed intervening sequences from Salmonella typhimurium.

We have introduced the intervening sequence (IVS) from 23S rRNA of the rrnD operon of Salmonella typhimurium into the equivalent position of Escherichia coli 23S rRNA. Salmonella typhimurium 23S rRNA is fragmented due to the RNase III-dependent removal of the approximately 100 nt stem-loop structure that comprises the IVS. In this study, we have found that insertion of the S. typhimurium IVS into E. coli 23S rRNA causes fragmentation of the RNA but does not affect ribosome function. Cells expressing the fragmented 23S rRNA exhibited wild-type growth rates. Fragmented RNA was found in the actively translating polysome pool and did not alter the sedimentation profile of ribosomal subunits, 70S ribosomes or polysomes. Finally, hybrid 23S rRNA carrying the A2058G mutation conferred high level erythromycin resistance indistinguishable from that of intact 23S rRNA carrying this mutation. These observations indicate that the presence of this IVS and its removal are phenotypically silent. As observed in an RNase III-deficient strain, processing of the IVS was not required for the production of functional ribosomes.     

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 ribosomal serine proteinase: cathepsin R.

As has been known for several years, thoroughly purified ribosomes contain a firmly bound serine proteinase with an optimum of activity at neutral pH. The present paper shows that the activity is found in free cytoplasmic ribosomes as well as in ribosomes detached from the membranes of the endoplasmic reticulum of rat liver. After ribosome dissociation, the proteinase activity is found only on the 40 S subunits. Recovery of the proteinase in the proteins of whole ribosomes or of 40 S subunits amounts to 44 and 65%, respectively. Ribosomes purified both from plant (Euglena) and bacterial (Acinetobacter) cells contain a serine proteinase having an activity quite comparable to that of rat liver ribosomes. In view of the recommendations of BARRETT et al. ( in REICH, RIFKIN and SHAW (eds).: Proteinases and Biological Control, Cold Spring Harbour Lab., 1975, p. 481), who no longer restrict the name "cathepsin" to acid or even lysosomal proteinases, we propose the name " ccathepsin R" for this ribosomal serine proteinase.     

On the rate of ribosome translocation anisotropy and ribosome saturation in the polysome

This study examines the rate of ribosome translocation in the mammalian polysome engaged in protein synthesis by utilizing our knowledge of the hydrodynamic behavior of the rat liver polysomes, sedimenting in a linear sucrose density gradient. The average distance between adjacent ribosomes in the polysome was estimated assuming an extended linear configuration of the polysomes during sedimentation. Based on this estimate, the velocity of ribosome movement along the messenger RNA appears to be non-uniform and inversely related to the ribosome content of the polysome. Such non-uniformity prevails at stages of translation prior to ribosome “saturation” of the polysome. A correlation has been made between the results reported herein and previously published evidence on the rate of polypeptide chain synthesis. The steady-state condition for the polypeptide chain assembly is viewed as representing the state of ribosome “saturation”, characterized by a minimal ribosome velocity and a maximum density of ribosome distribution, both functions being uniform throughout the entire length of the polysome.     

Dependence of the quality and the in vitro translation capacity of pine ribosome assemblies on the isolation procedure

The in vitro translation capacity of total ribosome assemblies isolated from the vegetative buds of small Scots pine (Pinus sylvestris L.) plants depends on the isolation procedure. Good yields and high values for protein synthesis were obtained in experiments in which polyvinyl pyrrolidone (PVP) was added to the grinding buffer. The polysome profiles obtained after sucrose density gradient centrifugation indicated the presence of polysomes in all samples. In addition, large ribosome aggregates were visible in the scanning electron micrographs. The use of an RNase inhibitor (RNasin) together with PVP did not improve the results, and treatment with ribonuclease (RNase, EC 3.1.27.5) destroyed the ability to synthesize protein. D, L-Dithiothreitol (DTT) and mercaptoethanol, if used instead of or together with PVP, gave low yields and also DTT destroyed the in vitro translation capacity of the ribosome assemblies. The polysome profiles had a high peak indicating dimers and often a descending series of peaks indicating polymers. A study of the scanning electron micrographs gave the impression that the largest polymers and aggregates had broken down. Protease K (EC 3.4.21.14) when added to the grinding buffer also destroyed the ability of the ribosomes to maintain protein synthesis in vitro. In this case, the shape of the polysome profiles gave the impression of successful isolation. Clumps of ribosomes, presumably originating from large aggregates, were visible in the scanning electron micrographs. Triton X-100 and 0.25 M NaCl in the grinding buffer extracted chromatin, which affected the results. The material lost during the extraction and purification processes consisted mainly of monosomes and their sub-units. On the basis of the above results it was concluded that the preservation of large polysomes and ribosome aggregates in the isolated ribosome assemblies is necessary if they are to maintain a high translation capacity. The content of the assemblies was best revealed in the scanning electron micrographs. The shape of the polysome profiles did not always correlate with the ability of the isolated ribosomes to synthesize proteins.     

Protein synthesis by microsomal particles from regenerating rat liver

1. Washed microsome particles from regenerating liver were shown to incorporate [(14)C]leucine into protein more actively than similar preparations from normal liver. 2. The total incorporation in the preparations from regenerating liver increased linearly with the amount of protein incubated, whereas this was not so with preparations from normal liver. 3. The greater activity of regenerating-liver microsomes appeared to be associated with the bound polysomes. 4. The size distribution of polysomes obtained after removal of membrane with deoxycholate was the same in normal and regenerating liver. 5. In general the activity of polysome preparations from normal and regenerating liver was similar. 6. It is concluded that the greater activity of the particles in the microsome fraction from regenerating liver is to be attributed to the ribosomes bound to membrane and that their activity is controlled by factors present in the membrane.     

Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [¹⁴C]- or [³H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·10⁴ and 2.1·10⁴ were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with $\text{N-[}{}^3\text{H]ethylmaleimide}$ or $\text{N-[}{}^{14}\text{C]ethylmaleimide}$, and the ³H to ¹⁴C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.     

Red-Far Red Reversible Effect on Polysome Formation in the Embryos of Pinus thunbergii Seeds

Polysome formation in the embryos of Pinus thunbergii seeds was studied. Free ribosomes were dissociated to smaller subunits in a high salt buffer, but the complex ribosomes were not. The free ribosomes could be distinguished from monomer ribosomes derived from polysomes after RNase treatment. The monomer ribosomes present in the embryos of the dark-imbibed seeds were predominantly free ribosomes; very small quantities of polysomes could be detected in the embryos from dark-imbibed seeds. Such polysomes remained at a very low level during dark imbibition at least for a month. The level of polysomes increased 4 hours after a brief exposure to red light. The effect of red light on polysome formation was partially reversed when followed by far red light irradiation.     

The isolation and properties of cardiac ribosomes and polysomes

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg²⁺ ions. The degradation in the absence of Mg²⁺ ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2·4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na⁺ and K⁺ ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.     

RNA processing enzymes RNase III, E and P in Escherichia coli are not ribosomal enzymes.

We recently showed that RNase III can process a small stable RNA, precursor 10Sa RNA, that accumulates in an rne (RNase E) strain at non-permissive temperatures. Precursor 10Sa (p10Sa) RNA is processed to 10Sa RNA in two steps, the first step is catalyzed by RNase III in the presence of Mn2+ but not Mg2+. It was shown that RNase III cosediments with membrane preparation from wild type as well as RNase III overexpressing cells. However, the possibility of membrane preparation contamination with ribosomes could not be ruled out. Here we show that RNase III, E and P are not associated with ribosomes. E. coli cells were opened either by alumina grinding or by sonication and fractionated into cytosolic and pellet fractions. The characterization of membrane preparations was done by assaying NADH oxidase, a bona fide membrane enzyme. Ribosomes prepared by alumina grinding were found to be contaminated with small fragments of membrane which contained RNase III activity. RNase III and NADH oxidase activities were present in the ribosomal preparations which could be solubilized by reagents that dissolve the inner membrane. Isopycnic sucrose gradient centrifugation of the membrane and ribosomal preparations also confirmed that RNase III fractionated with the inner membrane. Similarly RNase P activity was found in the corresponding fractions when isopycnic centrifugation of membrane and ribosome preparations was carried out. RNase E activity was also found to be present mostly in the post-ribosomal supernatant. These findings show that RNase III, E and P are not ribosomal enzymes.     

Batchwise Purification of Liver Ribosomes and Polysomes from Unfasted Mice on Hydroxylapatite

Batchwise purification of liver ribosomes and polysomes on hydroxyl-apatite is a rapid procedure to remove glycogen, hemoglobin, ribonuclease and other contaminants from ribosomal preparations. Ribosomes and polysomes are adsorbed to hydroxylapatite in a Büchner filter funnel and the contaminants are eluted from the hydroxylapatite with 0.15 M KH₂PO₄. The ribosomes and polysomes are then eluted with 0.3–0.4 M KH₂PO₄ and concentrated by centrifugation. The resolution of the polysome profiles was greatly improved following purification. The purified ribosomes could be dissociated into subunits at 0.3 M KCl, and showed no loss of activity in poly-U directed phenylalanine synthesis.