Hybrid Ribosomes Research Articles

Subunit homology between Escherichia coli, mitochondrial and chloroplast ribosomes

Interchange experiments between ribosomal subunits from spinach chloroplasts, yeast mitochondria and Escherichia coli have been per formed to obtain more information on possible homologies existing between them. Homology between bacterial and chloroplast ribosomes is high, since hybrid ribosomes containing chloroplast and E. coli subunits are active in polyphenylalanine synthesis directed by poly (U). Mitochondrial ribosomal subunits, in contrast, do not form hybrid ribosomes with either chloroplast or E. coli subunits.

Differences between ribosomal subunits from brain and those from other tissues

Ribosomal subunits were prepared from brain, liver and kidney of inbred mice by a process which includes treatment with deoxycholate and puromycin, and separation on 1.0 m-KCl gradients. The dependencies of reconstituted ribosomes of these tissues on magnesium and ammonium ions, and hybrids formed from subunits of differing tissues, were measured in poly(U)-directed polyphenylalanine synthesis. Brain ribosomes have a magnesium ion requirement that is twice as high as ribosomes from kidney and liver. Results with the hybrid ribosomes indicate that the 60 s subunit is responsible for this difference. Acrylamide gel electrophoresis of proteins from the subunits shows several significant differences between brain and liver ribosomes in both subunits. Control experiments failed to indicate that these results are due to supernatant contamination.

Rapid exchange of subunits between free ribosomes in extracts of Escherichia coli.

The runoff of a mixture of "heavy" and "light" polysomes has been reported to yield hybrid ribosomes, of intermediate density. However, we found that the dissociation of free heavy ribosomes as they advance in a sucrose gradient causes a serious artefact in the demonstration of hybrid ribosomes. This artefact is eliminated by fixation with glutaraldehyde before sedimentation. Formation of hybrid ribosomes can then be demonstrated, but it is equally complete whether the heavy and light preparations are mixed before or after runoff. Hence, free ribosomes exchange subunits rapidly in extracts. The previously reported exchange thus does not provide evidence for formation of free subunits during runoff.

Reaction of puromycin with N-acetylphenylalanyl-tRNA on ribosomes reassociated from escherichia coli ribosomal subunits

N-Acetylphenylalanylpuromycin synthesis catalyzed by ribosomes reassociated from 50-S and 30-S subunits was studied. N-Acetylphenylalanylpuromycin was formed even at o° by addition of puromycin to a mixture of 50-S ribosomal subunits and a preparation of 30-S ribosomal subunits, poly(U) and N-acetylphenylalanyl-tRNA previously mixed at o° after preincubation under suitable conditions. To obtain maximal formation of N-acetylphenylalanylpuromycin by reassociated ribosomes, the 50-S ribosomal subunits had to be preincubated at a sufficient concentration of NH 4 + (or K +) at a suitable temperature (about 37°), but a change in the concentration of Mg 2+ showed no significant effect. The preincubation of 30-S ribosomal subunits did not require a particular concentration of NH 4 + (or K +) but a sufficient Mg 2+ concentration was necessary. Using this technique we were able to study the effect of environmental conditions on the activity of respective ribosomal subunits in peptide synthesis. The 50-S and 30-S subunits preparations were obtained from ribosomes of parent Escherichia coli Q13 strain and from its mutant ribosomes which have an altered specific protein component in the 50-S subunit and a high monovalent cation (K + or NH 4 +) dependency in the peptidyl transfer reaction. The hybrid ribosomes consisted of Q13 30-S subunits and the mutant 50-S subunits showed distinctly higher monovalent cation dependency than the hybrid ribosomes from Q13 50-S subunits and the 30-S subunits of the mutant ribosomes, suggesting that alteration of 50-S ribosomal subunits induces alteration of their peptidyl transferase activity.

The protein-synthesizing activity of ribosomes isolated from the mammary gland of lactating and pregnant guinea pigs.

1. Polyribosome preparations were made from the deoxycholate-treated post-nuclear fractions obtained by the disruption of mammary glands from lactating and pregnant guinea pigs. 2. A high proportion of large polyribosomes was obtained from the glands of lactating animals whereas mainly small polyribosomes were obtained from the glands of pregnant animals. The isolated preparations incorporated [(14)C]phenylalanine into protein. The polyribosomes from the glands of pregnant animals were less active than those from the glands of lactating animals but the activity of the former was stimulated more by poly(U) than was the latter. 3. The ribosomes from mammary gland could be dissociated into subunits after incubation, under conditions necessary for protein synthesis, in the presence of puromycin. The subunits could be recombined to give a preparation that actively polymerized [(14)C]phenylalanine in the presence of poly(U). The subunits from guinea-pig mammary gland could be combined with subunits from liver of either guinea pig or rat. Hybrid ribosomes were also formed from subunits derived from glands of pregnant and lactating animals. The hybrids were as active as were the ribosomes formed by reassociation of subunits from the same tissue, suggesting that in this respect the ribosomes from pregnant animals were not defective. 4. Polyribosomes from mammary glands of lactating animals when incubated with cell sap from the same source were tested for their ability to synthesize alpha-lactalbumin. The polyribosomes were incubated in the presence of [(3)H]leucine and alpha-lactalbumin was isolated from the supernatant. The protein was finally treated with cyanogen bromide and the C-terminal and N-terminal fragments were separated and their radioactivity was determined. Both fragments were radioactive consistent with the synthesis of alpha-lactalbumin. 5. The results are discussed in relation to protein synthesis in the mammary gland after parturition.

Interaction of informational macromolecules with ribosomes: V. Reconstitution of specificity of ribosomes

1. 1. An attempt was made to reconstitute the ability of rat liver and thymus ribosomes to bind and recognize the homologous RNA messages. 2. 2. Protein factors detached from ribosomes by treatment with 1 M KCl are highly capable of binding RNA, but no specific recognition of homologous RNA by partially purified protein factors themselves was detected. 3. 3. Hybrid ribosomes reconstituted with particles and protein factors from rat liver and thymus ribosomes possessed properties of specificity similar to that of ribosomes from which protein factors are prepared. The possibility, that protein factors are really responsible for selective recognition of the particular species of RNA only when these factors are associated with particles was discussed.

Hybrid ribosome formation from Escherichia coli and chloroplast ribosome subunits.

A 70S ribosome was prepared from a 30S ribosome subunit from Euglena gracilis chloroplasts and a 50S ribosome subunit from Escherichia coli. This hybrid ribosome was active in polyuridylic acid-directed polyphenylalanine synthesis.

Properties of hybrid ribosomes formed from the subunits of mesophilic and thermophilic bacteria

The contributions of the individual ribosomal subunits to the thermostability of ribosomes in in vitro protein synthesis were examined by forming hybrid ribosomes from ribosomal subunits of the mesophilic bacterium Escherichia coli and the thermophilic bacterium Bacillus stearothermophilus. The relative stabilities of these hybrid ribosomes were determined by measuring their temperatures of maximum synthesis in the polyuridylic acid-phenylalanine-, polyadenylic acidlysine-, and polyuridylic-guanylic acid-valine-stimulated incorporating systems. In each of these amino-acid incorporating systems, the E. coli 30 s + B. stearothermophilus 50 s hybrid ribosome exhibited greater thermostability than the reciprocal hybrid ribosome. The thermostabilities of both hybrid ribosomes were greater in the poly (U,G)-valine system than in the other two systems. These latter results provide further evidence that messenger RNA copolymers containing purine residues increase the thermostability of the mRNA-ribosome-aminoacyl-transfer RNA complex. Physical and functional stabilities of individual ribosomal subunits were determined by ultraviolet thermal denaturation and thermal inactivation, respectively. By both criteria, the B. stearothermophilus 50 s ribosomal subunit was more thermostable than the E. coli 30 s ribosomal subunit by approximately 15 deg. C. The greater thermostability of the E. coli 30 s + B. stearothermophilus 50 s hybrid ribosome in these amino acid-incorporating systems is attributed to the presence of the more physically and functionally thermostable B. stearothermophilus 50 s ribosomal subunit. It is proposed that, at elevated temperatures, the B. stearothermophilus 50 s ribosomal subunit protects the E. coli 30 s ribosomal subunit from functional inactivation by helping to maintain its proper structural configuration.

Hybrid 80S monomers formed from subunits of ribosomes from protozoa, ungi, plants, and mammals.

Free 80S ribosomes of eukaryotic organisms are dissociated by KCl (0.8–1.0 m) in the presence of 2-mercaptoethanol and magnesium ions (10–15mm); the large and small subunits so formed can be recombined to yield 80S monomers. We have now studied the ability of ribosomal subunits from protozoa (Tetrahymena pyriformis), fungi (Allomyces arbuscula, Saccharomyces cerevisiae), plants (pea, wheat), and mammals (rat, mouse, rabbit) to combine to form hybrid ribosomes. In general, both subunits of the species studied participate in the formation of hybrid particles, with the exception of the 60S subunit of Tetrahymena, which does not combine with the small subunit of fungal, plant, or mammalian ribosomes. The interaction of subunits from rat and Tetrahymena ribosomes has been visualized by an electron microscope study of negatively stained preparations. The base sequences of the ribosomal RNAs of these organisms have been compared to those of Saccharomyces by nucleic acid hybridization-competition.

Functional capacity of intact and hybrid ribosomes from livers of normal and hypophysectomized rats

These studies were designed to determine whether liver ribosomes from normal and hypophysectomized rats are functionally equivalent. Monosomes derived from both sources, by disaggregation of polysomes, were found to be equally responsive to stimulation by polyuridylic acid (poly U) when incubated in a cell-free amino acid-incorporating system. Monosomes prepared by reassociation, in all possible combinations, of 40 and 60S subunits from normal and hypophysectomized rats again were equally active in the same system. This investigation suggests that liver ribosomes from normal and hypophysectomized rats are operationally identical.