Ribonuclease Activity Associated With Ribosomes of Zea mays

At pH 6.5, a ribonuclease(s) is associated with ribosomes isolated from corn (Zea mays L.) and cannot be removed by repeated differential centrifugation or by sedimenting through the sucrose gradient. The enzyme is active under conditions favoring the maintenance of integrity of the ribosomes. Little or no latent ribonuclease appears to be present. The activity of the enzyme at pH 5.8 is stimulated by KCl and inhibited by polyvinyl sulfate, zinc, and bentonite. Deoxyribonuclease is also found on the particles.

The enzyme can be removed from ribosomes by adsorption onto bentonite. Ribosomes are also adsorbed but to a much lesser extent at low bentonite concentrations. The enzyme is easily dissociated from ribosomes by raising the pH to 8.5, and readsorbed when the pH is lowered.

The ribonuclease activity on ribosomes shows a sharp increase with cell age that parallels closely the increase in total activity in the homogenate. The ratio of activities of deoxyribonuclease to ribonuclease on ribosomes also changes with cell age and again the changes appear to reflect changes in the homogenate. It is suggested that most of the association of ribonuclease with corn ribosomes may not be meaningful in vivo and occurs only after the cells are ruptured.

     

The Endoplasmic Reticulum of Mung Bean Cotyledons: ROLE IN THE ACCUMULATION OF HYDROLASES IN PROTEIN BODIES DURING SEEDLING GROWTH

The subcellular localization of two hydrolases (ribonuclease and vicilin peptidohydrolase) which are synthesized de novo in the cotyledons of mung bean seedlings was studied. Earlier experiments had shown that both enzymes accumulate in the protein bodies in the course of seedling growth. Two methods to fractionate subcellular organelles were used to demonstrate that a significant proportion of the enzymes is organelle-associated. This proportion is highest (up to 50% for vicilin peptidohydrolase and 15% for ribonuclease) when synthesis of the enzymes has just started. Evidence obtained with isopycnic sucrose gradients indicates that both hydrolases are associated with membranes rich in NADH-cytochrome c reductase, a marker enzyme for the endoplasmic reticulum (ER). The hydrolases band with the NADH-cytochrome c reductase under conditions where the ribosomes remain attached or are detached from the ER-derived vesicles. Treatment of the ER-derived vesicles with Triton X-100 shows that vicilin peptidohydrolase and vesicle membranes can be physically separated without dissolving the membranes, indicating that the proteinase is soluble within the vesicles. These data support the conclusion that the ER is involved in the transport of ribonuclease and proteinase to the protein bodies.     

The effect of pancreatic ribonuclease on rabbit reticulocyte ribosomes and its interpretation in terms of ribosome structure

1. Parts of the 16s and 30s RNA species of reticulocytes are readily hydrolysed by pancreatic ribonuclease. The biological activity of the ribosomes is diminished after treatment with low concentrations of the enzyme (e.g. 1ng. of ribonuclease/2.5mg. of polyribosome fraction/ml.). A high proportion of the chain scissions are ;hidden' owing to the secondary structure of the RNA moiety. 2. As the concentration of ribonuclease is increased RNA is lost from the ribosome. About 20-30% of the RNA may be removed from the ribosome without altering appreciably its sedimentation coefficient or its appearance in the electron microscope. 3. The amount of RNA removed from the ribosome is not increased by raising the concentration of enzyme from about 1mug. to 2.5mg. of ribonuclease/2.5mg. of polyribosome fraction/ml., or by increasing the temperature from 0 degrees to 30 degrees , or by first converting the RNA moiety into a single-stranded form before exposure to ribonuclease. 4. Untreated polyribosomes aggregate at about 75 degrees , whereas ribosomes treated with ribonuclease aggregate at about 45 degrees . The aggregates that are found on heating ribosomes after enzymic hydrolysis contain about 40-50% of the complement of RNA of intact ribosomes. 5. From the size of the fragments of RNA isolated from RNA-depleted ribosomes it is inferred that there is one site/60-100 nucleotides that is sensitive to ribonuclease. 6. The RNA moiety of RNA-depleted ribosomes has some double-helical character as shown by the optical properties and X-ray-diffraction pattern of ribonuclease-treated ribosomes and by the ;melting' properties of the isolated RNA. 7. Subparticles prepared by titration with an excess of EDTA are readily hydrolysed by ribonuclease to fragments of S(20,w) less than 4s, in contrast with the intact particle.     

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 Mg²⁺. 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.     

Autodegradation of RNA of plant cell ribosomes

The existence of an autodegradation capacity has been observed in the pith ribosomes of Nicotiana tabacum (L). This ribonucleolytic activity is activated fivefold by EDTA. The ribonuclease activity was also studied in resting and growing cells of pith blocks cultured on White's basic medium. Less ribonuclease activity was observed in rapidly dividing cells.     

Topography of the Escherichia coli 30 S ribosome revealed by the modification of ribosomal proteins

Chemical and enzymatic iodination were used to determine the position of proteins within the 30 S ribosome of Escherichia coli. The relative degree of iodination was determined in intact 30 S ribosomes, particles unfolded with EDTA, ribonuclease digests of ribosomes and extracted proteins. These procedures permitted an evaluation of the influence of protein-RNA and protein-protein interactions, protein conformation and position, on the degree to which protein could be modified by radioactive iodine. From the data we conclude that all 30 S ribosomal proteins are accessible to the external milieu, and that none are buried within the three-dimensional structure of the particle.     

The effect of ribonuclease on polysomes and ribosomes of bacteria and animal cells

The mildest treatment with ribonuclease that causes any disaggregation of the polysomes of Escherichia coli or HeLa cells simultaneously attacks the RNA of the constituent ribosomes. It is concluded that the susceptibility to ribonuclease of polysomes does not suggest that they are held together by a strand of messenger RNA. The RNA of the larger sub-unit of bacterial ribosomes has particularly sensitive regions resulting in a non-random degradation. The RNA of the smaller sub-unit of E. coli ribosomes is relatively resistant to ribonuclease attack. The same may be true of the respective sub-units of the intact HeLa-cell ribosome, but both sub-units become very sensitive to ribonuclease on dissociation from each other.     

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.     

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.     

Ribonuclease sensitivity of Escherichia coli ribosomes

Santer, Melvin (Haverford College, Haverford, Pa.), and Josephine R. Smith. Ribonuclease sensitivity of Escherichia coli ribosomes. J. Bacteriol. 92:1099-1110. 1966.-The ribonucleic acid (RNA) contained in 70S ribosomes and in 50S and 30S subunits was hydrolyzed by pancreatic ribonuclease. A 7% amount of the RNA was removed from the 70S particle; at 10(-4)m magnesium concentration, a maximum of 24 and 30% of the RNA in the 50S and the 30S fractions, respectively, was removed by ribonuclease. At the two lower magnesium ion concentrations, 50S ribosomes did not lose any protein, whereas 30S ribosomes lost protein as a result of ribonuclease treatment. A number of proteins were removed from the 30S particles by ribonuclease, and these proteins were antigenically related to proteins present in 50S ribosomes. The differential effect of ribonuclease on 50S and 30S ribosomes suggested that they have structural dissimilarities.     

Nuclease Activities of Mycoplasma gallisepticum as a Function of Culture Age in Different Media

Levels of deoxyribonuclease and ribonuclease activities in the supernatant (soluble plus ribosomes) fraction of Mycoplasma gallisepticum were assayed and found to be a function of strain, nutrient, and culture age. In yeast hydrolysate-enriched broth, maximal nuclease activities occurred during exponential growth.     

RIBOSOMES BOUND TO CHLOROPLAST MEMBRANES IN CHLAMYDOMONAS REINHARDTII

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein.     

Water Stress and Protein Synthesis: IV. RESPONSES OF A DROUGHT-TOLERANT PLANT

Responses of the drought-tolerant moss, Tortula ruralis, tosteady state water stress have been studied. A decrease in freshweight, an inhibition of amino acid incorporation into protein,and a decline in polysome population occur with increasing degreeof water stress. Cyclohexirnide treatment prevents the stress-inducedloss of polysomes. Ribonuclease activity increases during stressand this increase is partially prevented by cycloheximide. Thereis a lack of correlation between ribonuclease activity and polysomelevels. During stress, the decrease in polysome level and theincrease in ribonuclease activity do not coincide in time, theformer occurring earlier. Furthermore, ribosomes from the stressedmoss do not appear to be complexed with mRNA fragments, as indicatedby their inability to effect the formation of a peptide bond.It is concluded that the primary cause of polysome loss duringwater stress is the run-off of ribosomes from mRNA coupled withtheir failure to reinitiate.     

16s and 23s components in the ribosomal ribonucleic acid of Rhodopseudomonas spheroides

1. Ribosomal RNA was extracted from lysates of Rhodopseudomonas spheroides without prior isolation of ribosomes. 2. The composition of this RNA was investigated by using gradient centrifugation, showing that the proportion present as 23s component depended on the method of extraction. 3. The highest proportion of 23s component was found when cells were disrupted by ultrasonic treatment in the presence of ribonuclease inhibitors. 4. The results indicated that a ribonuclease is active in the cell lysate; this could account for the previous report (Lessie, 1965) that ribosomes of Rhodopseudomonas spheroides do not contain a 23s component.     

Polyribosomes from Pear Fruit: Changes during Ripening and Senescence

Polysome profiles were examined from lyophilized peel tissue of ripening pear (Pyrus communis, L. var. Passe-Crassane). Messenger RNA chains bearing up to eight ribosomes (octamers) were resolved and exhibited the highest absorption peak when ribonuclease activity was eliminated during extraction. Neither normal ripening nor the increase of large polyribosomes that normally accompanies ripening and senescence of the fruit occurred when pretreatment at 0 C was omitted. Normal ripening and increase of large polyribosomes would, however, be initiated by an ethylene treatment. The size distribution of the polyribosomes remained essentially constant throughout a 4-month cold storage; there was, however, a large increase in ribosomes by the 12th week of storage.     

The effect of ribonuclease on rat-liver ribosomes

1. Rat-liver ribosomes lose about 50% of their amino acid-incorporating activity when preincubated with ribonuclease. 2. This preincubation results also in loss of about 50% of the original protein content and 75% of the RNA. 3. Ribosomes sedimented by ultracentrifugation, after preincubation with ribonuclease, show negligible contamination by crystalline enzyme. 4. Washing of ribosomes treated with ribonuclease releases further protein, restoring the original RNA/protein ratio. 5. The washed particle is again capable of promoting amino acid incorporation. 6. Examination of ribosomes treated with ribonuclease in the analytical ultracentrifuge reveals destruction of ribosomes, disappearance of dimers and a decrease in the sedimentation coefficient of monomers. 7. Washed ribosomes consist of even smaller particles with a sedimentation coefficient 60s.     

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of ovariectomy and administration of oestradiol-17beta on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomal preparation

1. Three procedures for isolating ribonucleoprotein particles from the cytoplasmic fraction of rat-uterus homogenates are described. By procedure 1, ribonucleoprotein particles were isolated in the presence of 5mm-Mg²⁺ and 25mm-K⁺, and the postmitochondrial supernatant fraction was made to 1·3% (w/v) in potassium deoxycholate. About 50% of the RNA and protein of the microsomal fraction was recovered in the monomeric ribosomes isolated. By procedure 2, ribonucleoprotein particles were isolated in the presence of 10mm-Mg²⁺ and 0·1m-K⁺, and in the absence of detergent. The ribosomes obtained were primarily polymeric, but recovery of microsomal RNA and protein was only 32%. By procedure 3, ribonucleoprotein particles were isolated according to procedure 1 but without the use of detergent. A mixture of polymeric and monomeric ribosomes was obtained, and the recovery of microsomal RNA and protein was about 60%. 2. Uterine polymeric and monomeric ribosomes, isolated by procedure 3 and designated `polyribosomal preparation', were examined for protein-synthesizing capabilities. The principal properties of the cell-free protein-synthesizing system containing the polyribosomal preparation are described. The efficiency of amino acid incorporation in the complete system incubated for 30min. and containing the polyribosomal preparation was found to be either 2·5 molecules of [¹⁴C]leucine or 2·2 molecules of [¹⁴C]-valine incorporated/ribosome. Assay of the preparation in the complete cell-free system containing 10mm-sodium fluoride indicated that 40% of the incorporation activity is a result of initiation of new polypeptide chains and 60% is due to completion of previously existing chains. Monomeric ribosomes obtained by various treatments of the polyribosomal preparation with sodium fluoride, ribonuclease and potassium deoxycholate had decreased incorporation activity in the cell-free system. However, monomeric ribosomes obtained by treatment with sodium fluoride only had an incorporation activity 50% greater than that of monomers obtained by treatment with ribonuclease only. 3. The results indicate that uterine polymeric and monomeric ribosomes are sites of amino acid incorporation in vivo and in vitro. It is concluded that most polymeric and monomeric ribosomes occurring in the cytoplasmic fraction of the uterus are free and unattached to membranes, and that the polyribosomes are relatively unstable.     

The composition of an unusual precursor of 50 S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15--28 is a mutant which during exponential growth contains large amounts of a '47S' ribonucleoprotein precursor to 50S ribosomes. The '47S particles' are more sensitive to ribonuclease than are 50S ribosomes. The 23 S RNA of 47S particles may be slightly undermethylated, but cannot be distinguished from the 23S RNA of 50S ribosomes by sedimentation or electrophoresis. Isolated particles have 10--15% less protein than do 50S ribosomes; proteins L16, L28 and L33 are absent. Comparison with precursor particles studied by other workers in wild-type strains of E. coli suggests that the assembly of 50S ribosomes in strain 15--28 is atypical.     

The conformation of ribonucleic acids in Escherichia coli ribosomes: Inferences from the mode of action of ribonuclease II

1. Ribonuclease II of Escherichia coli degrades pulse-labelled RNA associated with ribosomes and polyuridylic acid on ribosomes and in solution to mononucleotides. 2. Ribosomal and pulse-labelled RNA in solution and ribosomal RNA in chloramphenicol particles (protein-deficient ribosomes) are degraded to oligonucleotides. 3. Ribosomal RNA in mature ribosomes is not attacked by the enzyme. 4. From the mode of action of ribonuclease II, which is specific for single-stranded polyribonucleotides and does not attack helical forms, it is inferred that pulse-labelled RNA associated with ribosomes of E. coli exists as a single-stranded structure and that ribosomal RNA in chloramphenicol particles has a pronounced helical character. 5. The different behaviour of ribonuclease II towards newly synthesized RNA, ribosomal RNA and chloramphenicol-particle RNA in E. coli ribosomes is discussed.     

Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii.

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.