The effect of ribonuclease on polysomes and ribosomes of bacteria and animal cells.
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ABSTRACT:
SUBMITTER: Fenwick ML
PROVIDER: S-EPMC1198691 | biostudies-other | 1968 Apr
REPOSITORIES: biostudies-other
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Project description:1. The ;30s' and ;50s' ribosomes from ribonuclease-active (Escherichia coli B) and -inactive (Pseudomonas fluorescens and Escherichia coli MRE600) bacteria have been studied in the ultracentrifuge. Charge anomalies were largely overcome by using sodium chloride-magnesium chloride solution, I 0.16, made 0-50mm with respect to Mg(2+). 2. Differentiation of enzymic and physical breakdown at Mg(2+) concentrations less than 5mm was made by comparing the properties of E. coli B and P. fluorescens ribosomes. 3. Ribonuclease-active ribosomes alone showed a transformation of ;50s' into 40-43s components. This was combined with the release of a small amount of ;5s' material which may be covalently bound soluble RNA. Other transformations of the ;50s' into 34-37s components were observed in both ribonuclease-active and -inactive ribosomes at 1.0-2.5mm-Mg(2+), and also with E. coli MRE600 when EDTA (0.2mm) was added to a solution in 0.16m-sodium chloride. 4. Degradation of ribonuclease-active E. coli B ribosomes at Mg(2+) concentration 0.25mm or less was coincident with the formation of 16s and 21s ribonucleoprotein in P. fluorescens, and this suggested that complete dissociation of RNA from protein was not an essential prelude to breakdown of the RNA by the enzyme. 5. As high Cs(+)/Mg(2+) ratios cause ribosomal degradation great care is necessary in the interpretation of equilibrium-density-gradient experiments in which high concentrations of caesium chloride or similar salts are used. 6. The importance of the RNA moiety in understanding the response of ribosomes to their ionic environment is discussed.
| S-EPMC1207202 | biostudies-other
Project description: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.
| S-EPMC1198493 | biostudies-other
Project description: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 Mg(2+) ions. The degradation in the absence of Mg(2+) 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.
| S-EPMC1206608 | biostudies-other
Project description:The ribosome is an essential cellular machine performing protein biosynthesis. Its structure and composition are highly conserved in all species. However, some bacteria have been reported to have an incomplete set of ribosomal proteins. We have analyzed ribosomal protein composition in 214 small bacterial genomes (<1 Mb) and found that although the ribosome composition is fairly stable, some ribosomal proteins may be absent, especially in bacteria with dramatically reduced genomes. The protein composition of the large subunit is less conserved than that of the small subunit. We have identified the set of frequently lost ribosomal proteins and demonstrated that they tend to be positioned on the ribosome surface and have fewer contacts to other ribosome components. Moreover, some proteins are lost in an evolutionary correlated manner. The reduction of ribosomal RNA is also common, with deletions mostly occurring in free loops. Finally, the loss of the anti-Shine-Dalgarno sequence is associated with the loss of a higher number of ribosomal proteins.
| S-EPMC7782861 | biostudies-literature
Project description:1. A comparison has been made between the ribonuclease activities of untreated ribosomes from Escherichia coli B and Pseudomonas fluorescens and the activities of ribosomes on to which ribosomal ribonuclease from E. coli B has been adsorbed. 2. The normal ribosomes from both species were stable in 5-10mm-Mg(2+) (I0.16) at pH6. The RNA in ribosomes from Ps. fluorescens was attacked by the adsorbed ribonuclease under these conditions, whereas the ribosomes from E. coli B were able to adsorb and inhibit this enzyme. 3. Inhibition was also observed with ribosomes from Aerobacter aerogenes, Proteus vulgaris and two other strains of E. coli. It was not observed in ribosomes from three species of Pseudomonas. 4. The inhibition depended on the integrity of the ribosomes and was not observed under conditions of low Mg(2+) concentration that cause irreversible degradation into more slowly sedimenting particles.
| S-EPMC1264755 | biostudies-other
Project description:Starvation of rats for several days led to marked decrease in cytoplasmic polysomes and accumulation of breakdown products having S values less than 200s. Re-feeding of the starved animals induced a rapid reassembly of polysomes. These newly formed polysomes, in the presence of actinomycin D, decayed in a biphasic fashion: about two-thirds decayed with an apparent half-life of 3-3(1/2)hr. but the other one-third were much more stable. Evidence that polysome decay is an accurate reflexion of messenger RNA stability is presented, and it is concluded that in the presence of large doses of actinomycin D, rat-liver cytoplasm contains messenger RNA classes of widely varying stability, the more stable class having a half-life of at least 80hr. The half-life of liver ribosomes was also determined and was found to be 110-127hr.
| S-EPMC1270442 | biostudies-other
Project description: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.
| S-EPMC1184962 | biostudies-other
Project description:1. Treatment of rat liver polysomes in a buffer containing 2.5mm-magnesium chloride with T(1) ribonuclease at a concentration of 330units/ml. of reaction medium at 37 degrees for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of (3)H-labelled polysomes (structural RNA labelled with orotic acid) with T(1) ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying (14)C-labelled nascent peptide chains with T(1) ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0.48mumole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying (14)C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0.5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying (14)C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride, followed by addition of transfer RNA as marker and chromatography on Sephadex G-200, show in both cases that considerable amounts of labelled peptide material move well ahead of the added transfer RNA marker. Further, if nucleoprotein particles carrying labelled peptide chains are treated with 0.3m-potassium hydroxide at 20 degrees for 24 hr., neutralized to pH7.6, and then chromatographed on Sephadex G-200, the labelled peptide material moves much closer to the added transfer RNA marker. These results suggest that a proportion of the nascent (14)C-labelled peptides on the nucleoprotein are attached to transfer RNA or large fragments of transfer RNA. 6. [(3)H]Polyuridylic acid binds to nucleoprotein particles in 1mm-magnesium chloride. The rate of binding is rapid when measured at 20 degrees .
| S-EPMC1184744 | biostudies-other
Project description:Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and translational control and its interaction with ribosomal drugs. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. To study the interaction of aminoglycoside antibiotics with selected eukaryotic ribosomes, we replaced the bacterial drug binding site in 16S rRNA with its eukaryotic counterpart, resulting in bacterial hybrid ribosomes with a fully functional eukaryotic rRNA decoding site. Cell-free translation assays demonstrated that hybrid ribosomes carrying the rRNA decoding site of higher eukaryotes show pronounced resistance to aminoglycoside antibiotics, equivalent to that of rabbit reticulocyte ribosomes, while the decoding sites of parasitic protozoa show distinctive drug susceptibility. Our findings suggest that phylogenetically variable components of the ribosome, other than the rRNA-binding site, do not affect aminoglycoside susceptibility of the protein-synthesis machinery. The activities of the hybrid ribosomes indicate that helix 44 of the rRNA decoding site behaves as an autonomous domain, which can be exchanged between ribosomes of different phylogenetic domains for study of function.
| S-EPMC2094070 | biostudies-literature
Project description:The catalytic RNA component of bacterial RNase P is responsible for the removal of 5' leader sequences from precursor tRNAs. As part of an on-going phylogenetic comparative characterization of bacterial RNase P, the genes encoding RNase P RNA from the thermophiles Thermotoga maritima, Thermotoga neapolitana, Thermus aquaticus, and a mesophilic relative of the latter, Deinococcus radiodurans, have been cloned and sequenced. RNAs transcribed from these genes in vitro are catalytically active in the absence of other components. Active holoenzymes have been reconstituted from the T.aquaticus and T.maritima RNAs and the protein component of RNase P from Escherichia coli. The RNase P RNAs of T.aquaticus and T.martima, synthesized in vitro, were characterized biochemically and shown to be inherently resistant to thermal disruption. Several features of these RNAs suggest mechanisms contributing to thermostability. The new sequences provide correlations that refine the secondary structure model of bacterial RNase P RNA.
| S-EPMC309168 | biostudies-other