Messenger RNA turnover and protein synthesis in B. subtilis inhibited by actinomycin D (original) (raw)
1962, … of Sciences of the United States …
The hypothesis that protein synthesis in bacteria is mediated by an unstable, rapidly turning over RNA fraction, designated messenger RNA (mRNA),' is well supported by evidence from a variety of experiments. When either phage-in-fected2-4 or normal5' 6 bacterial cells are given short pulses of radioactive RNA precursors, the radioactivity is incorporated into an unstable RNA having a base composition similar to that of DNA and able to form molecular hybrids with it.' This RNA fraction is distinguishable from the ribosomal and transfer RNA by its sedimentation behavior and by its ability to stimulate protein synthesis in cell-free ribosomal systems.8 9
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Proceedings of The National Academy of Sciences, 1962
Actinomycin D is one of a number of polypeptide antibiotics isolated in Waksman's laboratory.1' 2 Bacteriostatic effects, particularly on gram positive bacteria, and antitumor activity have been attributed to this compound.2 3 Kirk4 has demonstrated that the addition of actinomycin D (0.2 to 0.5 ,4M) to exponentially growing cultures of Staphylococcus aureus stops RNA synthesis immediately. This effect is rapidly followed by an inhibition of protein synthesis, and later by a partial inhibition of DNA synthesis. The action of this compound is not related directly to energy production since both respiration and glycolysis of inhibited cells are unaffected by concentrations up to 0.1 mM.4 Kirk also demonstrated that the combination of DNA and actinomycin D results in a spectral change of the latter compound. These observations suggest the formation of a complex between these two compounds since Kawamata and Imanishi5 found no interaction of actinomycin and RNA and the reaction appears to be relatively specific for DNA. Although Rauen et al.6 have reported complex formation between actinomycin and RNA, 100 times more RNA than DNA is required.
Journal of General Virology, 1979
Cytoplasmic RNA synthesis can be detected in vaccinia virus-infected HeLa cells in the presence of 2/zg/ml but not 2o/*g/ml of actinomycin D. When RNA synthesis is observed protein synthesis is inhibited in infected, treated cells. We had previously noted that such a correlation may also be observed in infected, cycloheximide-treated cells. If actinomycin D (20/zg/ml) is added to these cells at various times after infection and treatment, the inhibition of protein synthesis seen upon removal of cycloheximide does not continue beyond the point to which it had developed before the actinomycin D was added. These results indicate that the inhibition of protein synthesis can be correlated with the amount of cytoplasmic RNA synthesized in infected cells and that this RNA synthesis and the subsequent inhibition of protein synthesis can be prevented by sufficiently high concentrations of actinomycin D. The cytoplasmic RNA which is synthesized does not appear to consist of double-stranded RNA nor of extensive self complementary regions. The cytoplasmic RNA synthesized in infected, cycloheximide treated cells appears to consist of early virus mRNA which can function as mRNA in vitro in a cell-free system derived from normal cells. An examination of the phosphorylation of ribosomal proteins shows six additional phosphoproteins in infected cells, two of which may be observed in infected cycloheximide-treated ceils, suggesting that phosphorylation of ribosomal proteins cannot be directly correlated with the inhibition of overall protein synthesis seen in infected cycloheximide-treated cells.
European Journal of Biochemistry, 1978
Cytoplasmic polyadenylated RNA of myogenic cells was shown to decay with biphasic kinetics, suggesting the existence of two main populations of mRNA with respect to stability. In the present study, the stability of mRNA extracted from actinomycin-D-treated cultures of a myogenic cell line was tested by its capacity to direct protein synthesis in the wheat germ cell-free system. The products were analyzed by dodecylsulphate/polyacrylamide gel electrophoresis. All major radioactive bands found in gels used for analyzing the products of the cell-free system directed by polyadenylated RNA extracted from untreated cultures were also found in similar gels containing products of RNA extracted after many hours of application of actinomycin D. The capacity to code for specific protein bands decays with a half-life ranging between 11 and 40 h. No fast-decaying translatable mRNA could be detected by this method. Instead, it was found that during the first 4-6 h following application
Site of actinomycin-resistant RNA synthesis in animal cells
Experimental Cell Research, 1973
RNA synthesized in animal cells in the presence of high doses of actinomycin had many of the properties associated with hnRNA. The nucleoplasm was established as the site of this synthesis, as determined by radioautography and by differential extraction. No synthesis was observed in nucleoli. Therefore this RNA, which was unmethylated, did not represent abortively synthesized incomplete ribosomal RNA chains. Resistance to ethidium bromide also ruled out mitochondria as the site of synthesis of this RNA. RNA synthesized in the presence of actinomycin like hnRNA was resistant to inhibition by cordycepin. RNA synthesized in the presence of actinomycin contained a ribonuclease-resistant core. When first synthesized this RNA was associated with a high molecular weight RNA which was degraded rapidly. This unique species of RNA synthesized in the nucleoplasm of animal cells may exert a control function in the transcription of hnRNA in the cell nucleus or may play a role in the transport of mRNA from nucleus to cytoplasm.
Microbiology, 1982
Cultures were labelled with ~-[~~S]methionine for 5 min periods immediately after resuspension in sporulation medium (to) or at hourly intervals thereafter (t l , t2 or t3). Cells were harvested and lysed very rapidly at low temperature in the presence of inhibitors of protease, cell extracts were subjected to high-resolution two-dimensional gel electrophoresis, and radioactive proteins were revealed by fluorography. Comparisons of fluorograms from wild-type cells labelled at various times after resuspension showed that 35 proteins were synthesized at t l that were not seen at to, ten at t2 that were not seen at tl, and seven at t3 that were not seen at t Z. Conversely, nine proteins ceased to be synthesized in the first hour of resuspension, eight in the second hour and five in the third hour. Experiments with the same protocol were done with cells of a strain carrying spo-43, a mutation that blocks sporulation at a very early stage. The results show that the mutation prevents about two-thirds of the total number of changes that occur in the wildtype in the first hour, about one-third of those that occur in the second hour, but only two out of 12 of those that occur in the third hour. METHODS Murerials. Ampholines were from LKB. Deoxyribonuclease I (pancreatic, chromatographically purified), ribonuclease A (pancreatic, 5 x crystallized) and lysozyme (Grade I) were from Sigma. 2-Mercaptoethanol and urea were from Bio-Rad as were acrylamide and the other materials necessary for gel electrophoresis. EDTA was neutralized with NaOH. ~-[~~S]Methionine [ > 1000 Ci (37 TBq) mmol-'1 was from Amersham.
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