Properties of 70S and 80S ribosomes from tobacco leaves

1. The interaction of exogenous messengers with 70 <em>S</em> chloroplast and 80 <em>S</em> cytoplasmic tobacco ribosomes in vitro was studied. The isolation of an amino acid incorporating system from tobacco leaves should enable us to study some aspects of viral protein synthesis. Tobacco 70 <em>S</em> and 80 <em>S</em> ribosomes were chosen for the following reasons: Little is known about the interaction of messenger RNA with 80 <em>S</em> ribosomes. In general, plant viral RNAs did not yield meaningful results in the <em>E. coli</em> system. The interaction of 70 <em>S</em> and 80 <em>S</em> ribosomes with messengers could be compared in a homologous tobacco leave system.2. The amino acid incorporating system as isolated initially could be characterized by its dependence on an energy source and GTP, on the concentration of ribosomes, on the magnesium and potassium ion concentration, and on the incubation time, but not on tRNA and soluble enzymes or on unlabeled amino acids.3. The lack of an effect of tRNA or soluble enzymes was not due to the amino acid activation reaction, but to the presence of these factors in the ribosome preparations.4. The ribosomes could be purified by sucrose gradient centrifugation, centrifugation through 0.5 M NH ₄ Cl, and by chromatography on a Sephadex G200 column. Then amino acid incorporation became dependent on the addition of tRNA and soluble enzymes but not of ¹²C-amino acids. It was calculated that even after purification about 0.0012 μmoles ¹²C-amino acids were still present in the ribosome preparation.5. Highly purified ribosomes were active for a longer period than nonpurified ribosomes, but nevertheless the incorporation rate decreased during incubation. The conclusion was drawn that both degradation and read out of the messenger were responsible for this decrease.6. Some of the physical properties of 70 <em>S</em> and 80 <em>S</em> ribosomes were determined. Methods were developed to separate 70 <em>S</em> and 80 <em>S</em> ribosomes and polyribosomes from each other. The polyribosomes appeared to consist of 80 <em>S</em> ribosomes. About 5-10 times as much 80 <em>S</em> ribosomes could be isolated from leaves as 70 <em>S</em> ribosomes.7. The 70 <em>S</em> ribosomes were not stable at elevated salt concentrations. At low Mg ²⁺concentrations they dissociated reversibly into 50 <em>S</em> and 35 S subunits; 80 <em>S</em> ribosomes could not dissociate, but unfolded at low Mg ²⁺concentrations. During this process 60 <em>S</em> particles first appeared, followed by 50 <em>S</em> and 40 <em>S</em> particles. When the Mg ²⁺concentration was raised again, 80 <em>S</em> particles reformed from 60 <em>S</em> particles but hardly from 50 <em>S</em> and 40 S particles.8. Both 70 <em>S</em> and 80 <em>S</em> ribosomes contained two different RNA molecules, i.e., the 70 <em>S</em> ribosomes had 17 <em>S</em> and 23 <em>S</em> RNA, and the 80 <em>S</em> ribosomes 17 Sand 25 <em>S</em> RNA.9. The amino acid incorporating activity of the 70 <em>S</em> and the 80 <em>S</em> ribosomes as compared to that of the polyribosomes was not essentially different, but the 70 <em>S</em> ribosomes were about 2.5 times as active as the 80 <em>S</em> ribosomes at their optimal Mg ²⁺concentrations. The optimal concentration for the 80 <em>S</em> ribosomes was 5 mM and for the 70 <em>S</em> ribosomes 17 mM.10. In contrast to the 80 <em>S</em> ribosomes, the 70 <em>S</em> ribosomes had a constant incorporation rate for about 40 minutes, which then decreased abruptly to almost zero. It was concluded that no messenger degradation took place during incubation, but that the messengers had been read out after 40 minutes. The 80 <em>S</em> ribosomes behaved similarly as mentioned for the mixture of ribo somes.11. Incubation temperature and pH had a clear effect on both 70 <em>S</em> and 80 <em>S</em> ribosomes. This effect, however, was variable and depended on the in cubation time. In general, the optimum temperature and pH decreased with increasing periods of incubation.12. The effect of exogenous messengers on the amino acid incorporation was studied. Untreated ribosomes could hardly be stimulated by such messen gers.13. Two methods were developed for the elimination of endogenous messen gers from 80 <em>S</em> ribosomes. According to the first method 80 <em>S</em> ribosomes were preincubated at pH 8.6 in a complete, but unlabeled, incubation mixture for 3 hours. Then the ribosomes were pelleted at 105,000 x g and incubated in a complete labeled incubation mixture. The second method consisted of dialysis against 10 ^-4M Mg ²⁺in standard buffer during 14 hours. During this dialysis the ribosomes unfolded to 60 <em>S</em> particles. The dissociation of 70 <em>S</em> ribosomes into 50 <em>S</em> and 35 <em>S</em> subunits proved to result in the elimination of endogenous messengers from these ribosomes.14. Preincubated and dialyzed 80 <em>S</em> and 70 <em>S</em> ribosomes could be programmed by poly U as an exogenous messenger. Incorporation continued at a constant rate for over 2 hours. The optimal Mg ²⁺concentrations were I11 mM for the 80 <em>S</em> and 18 mM for the 70 <em>S</em> ribosomes, respectively. At these concentrations, a stimulation of 10-40 times was obtained. Again, the 70 <em>S</em> ribosomes were about 2.5 times as active as the 80 <em>S</em> ribosomes. The activities were 30-45 μμmoles phenylalanine per mg per hour for the 80 <em>S</em> ribosomes and 70-140 μμmoles for the 70 <em>S</em> ribosomes.15. Viral RNAs did not stimulate to an appreciable extent the amino acid incorporation by either type of ribosomes. Initiation factors such as leucovorin were added, but none of these showed any effect. Some fractions, isolated from the supernatant of ribosomes centrifuged at 105,000 x g however, showed a small effect on the over-all incorporation. It was concluded that in our system some special initiation factor(s) may be missing and that it might be possible to isolate from the supernatant fractions such a factor or factors.