Abstract
Polysome analysis has proved to be a sensitive probe for the mode of action of inhibitors of protein synthesis in intact HeLa cells. To classify the active compounds as inhibitors of initiation, elongation, or termination, their effects on the cellular polyribosome pattern were compared under three conditions. These conditions tested (i) their direct effect on the polyribosome profile; (ii) their effect on ribosome run-off produced by hypertonicity; and (iii) their effects on recovery from hypertonicity. Using this technique, diacetoxyscirpenol, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and three alkaloids, harringtonine, isoharringtonine, and homoharringtonine, were found to be inhibitors of initiation. Polysome analysis indicated that in HeLa cells 7.8 × 10−7 M pactamycin, which inhibited protein synthesis 94%, interfered with elongation as well as initiation under these conditions. Emetine, anisomycin, cycloheximide, and trichodermin each gave polysome patterns consistent with inhibition of elongation. Fusidic acid and aurintricarboxylic acid inhibited incorporation of [14C]leucine into intact HeLa cells, but polysome analysis did not localize any specific inhibitory effects to the initiation, elongation, or termination steps of protein synthesis. The use of specific inhibitors of initiation of protein synthesis has indicated that most, if not all, mammalian messenger ribonucleic acids contain a single initiation site.
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Selected References
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- Baltimore D., Jacobson M. F., Asso J., Huang A. S. The formation of poliovirus proteins. Cold Spring Harb Symp Quant Biol. 1969;34:741–746. doi: 10.1101/sqb.1969.034.01.083. [DOI] [PubMed] [Google Scholar]
- Butterworth B. E., Rueckert R. R. Gene order of encephalomyocarditis virus as determined by studies with pactamycin. J Virol. 1972 May;9(5):823–828. doi: 10.1128/jvi.9.5.823-828.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carrasco L., Barbacid M., Vazquez D. The trichodermin group of antibiotics, inhibitors of peptide bond formation by eukaryotic ribosomes. Biochim Biophys Acta. 1973 Jun 23;312(2):368–376. doi: 10.1016/0005-2787(73)90381-x. [DOI] [PubMed] [Google Scholar]
- Cundliffe E. Antibiotics and polyribosomes. II. Some effects of lincomycin, spiramycin, and streptogramin A in vivo. Biochemistry. 1969 May;8(5):2063–2066. doi: 10.1021/bi00833a042. [DOI] [PubMed] [Google Scholar]
- Cundliffe E., Cannon M., Davies J. Mechanism of inhibition of eukaryotic protein synthesis by trichothecene fungal toxins. Proc Natl Acad Sci U S A. 1974 Jan;71(1):30–34. doi: 10.1073/pnas.71.1.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
- EAGLE H. Amino acid metabolism in mammalian cell cultures. Science. 1959 Aug 21;130(3373):432–437. doi: 10.1126/science.130.3373.432. [DOI] [PubMed] [Google Scholar]
- Ennis H. L. Polysome metabolism in Escherichia coli: effect of antibiotics on polysome stability. Antimicrob Agents Chemother. 1972 Mar;1(3):197–203. doi: 10.1128/aac.1.3.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grollman A. P., Huang M. T. Inhibitors of protein synthesis in eukaryotes: tools in cell research. Fed Proc. 1973 Jun;32(6):1673–1678. [PubMed] [Google Scholar]
- Grollman A. P. Inhibitors of protein biosynthesis. II. Mode of action of anisomycin. J Biol Chem. 1967 Jul 10;242(13):3226–3233. [PubMed] [Google Scholar]
- Grollman A. P. Inhibitors of protein biosynthesis. V. Effects of emetine on protein and nucleic acid biosynthesis in HeLa cells. J Biol Chem. 1968 Aug 10;243(15):4089–4094. [PubMed] [Google Scholar]
- Lodish H. G., Housman D., Jacobsen M. Initiation of hemoglobin synthesis. Specific inhibition by antibiotics and bacteriophage ribonucleic acid. Biochemistry. 1971 Jun 8;10(12):2348–2356. doi: 10.1021/bi00788a027. [DOI] [PubMed] [Google Scholar]
- Macdonald J. S., Goldberg I. H. An effect of pactamycin on the initiation of protein synthesis in reticulocytes. Biochem Biophys Res Commun. 1970 Oct 9;41(1):1–8. doi: 10.1016/0006-291x(70)90460-2. [DOI] [PubMed] [Google Scholar]
- McKeehan W., Hardesty B. The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulocytes. Biochem Biophys Res Commun. 1969 Aug 15;36(4):625–630. doi: 10.1016/0006-291x(69)90351-9. [DOI] [PubMed] [Google Scholar]
- Morrison T. G., Lodish H. F. Translation of bacteriophage Q RNA by cytoplasmic extracts of mammalian cells. Proc Natl Acad Sci U S A. 1973 Feb;70(2):315–319. doi: 10.1073/pnas.70.2.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pestka S. Antibiotics as probes of ribosome structure: binding of chloramphenicol and erythromycin to polyribosomes; effect of other antibiotics. Antimicrob Agents Chemother. 1974 Mar;5(3):255–267. doi: 10.1128/aac.5.3.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pestka S., Hintikka H. Studies on the formation of ribonucleic acid-ribosome complexes. XVI. Effect of ribosomal translocation inhibitors on polyribosomes. J Biol Chem. 1971 Dec 25;246(24):7723–7730. [PubMed] [Google Scholar]
- Pestka S. Inhibitors of ribosome functions. Annu Rev Microbiol. 1971;25:487–562. doi: 10.1146/annurev.mi.25.100171.002415. [DOI] [PubMed] [Google Scholar]
- Pestka S. Studies on transfer ribonucleic acid-ribosome complexes. XIX. Effect of antibiotics on peptidyl puromycin synthesis on polyribosoms from Escherichia coli. J Biol Chem. 1972 Jul 25;247(14):4669–4678. [PubMed] [Google Scholar]
- Pestka S. The use of inhibitors in studies on protein synthesis. Methods Enzymol. 1974;30:261–282. doi: 10.1016/0076-6879(74)30030-4. [DOI] [PubMed] [Google Scholar]
- Petersen N. S., McLaughlin C. S. Monocistronic messenger RNA in yeast. J Mol Biol. 1973 Nov 25;81(1):33–45. doi: 10.1016/0022-2836(73)90245-3. [DOI] [PubMed] [Google Scholar]
- Powell R. G., Weisleder D., Smith C. R., Jr Antitumor alkaloids for Cephalataxus harringtonia: structure and activity. J Pharm Sci. 1972 Aug;61(8):1227–1230. doi: 10.1002/jps.2600610812. [DOI] [PubMed] [Google Scholar]
- Robbins E., Pederson T., Klein P. Comparison of mitotic phenomena and effects induced by hypertonic solutions in HeLa cells. J Cell Biol. 1970 Feb;44(2):400–416. doi: 10.1083/jcb.44.2.400. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saborio J. L., Pong S. S., Koch G. Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J Mol Biol. 1974 May 15;85(2):195–211. doi: 10.1016/0022-2836(74)90360-x. [DOI] [PubMed] [Google Scholar]
- Schindler D. Two classes of inhibitors of peptidyl transferase activity in eukaryotes. Nature. 1974 May 3;249(452):38–41. doi: 10.1038/249038a0. [DOI] [PubMed] [Google Scholar]
- Schreier M. H., Staehelin T., Gesteland R. F., Spahr P. F. Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system. J Mol Biol. 1973 Apr 15;75(3):575–578. doi: 10.1016/0022-2836(73)90462-2. [DOI] [PubMed] [Google Scholar]
- Stafford M. E., McLaughlin C. S. Trichodermin, a possible inhibitor of the termination process of protein synthesis. J Cell Physiol. 1973 Aug;82(1):121–128. doi: 10.1002/jcp.1040820114. [DOI] [PubMed] [Google Scholar]
- Taber R., Rekosh D., Baltimore D. Effect of pactamycin on synthesis of poliovirus proteins: a method for genetic mapping. J Virol. 1971 Oct;8(4):395–401. doi: 10.1128/jvi.8.4.395-401.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tai P. C., Wallace B. J., Davis B. D. Actions of aurintricarboxylate, kasugamycin, and pactamycin on Escherichia coli polysomes. Biochemistry. 1973 Feb;12(4):616–620. doi: 10.1021/bi00728a008. [DOI] [PubMed] [Google Scholar]
- Tanaka N., Kinoshita T., Masukawa H. Mechanism of protein synthesis inhibition by fusidic acid and related antibiotics. Biochem Biophys Res Commun. 1968 Feb 15;30(3):278–283. doi: 10.1016/0006-291x(68)90447-6. [DOI] [PubMed] [Google Scholar]
- Tate W. P., Caskey C. T. Peptidyltransferase inhibition by trichodermin. J Biol Chem. 1973 Nov 25;248(22):7970–7972. [PubMed] [Google Scholar]
- Weeks D. P., Baxter R. Specific inhibition of peptide-chain initiation by 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide. Biochemistry. 1972 Aug 1;11(16):3060–3064. doi: 10.1021/bi00766a018. [DOI] [PubMed] [Google Scholar]
- Wei C. M., Hansen B. S., Vaughan M. H., Jr, McLaughlin C. S. Mechanism of action of the mycotoxin trichodermin, a 12,13-epoxytrichothecene. Proc Natl Acad Sci U S A. 1974 Mar;71(3):713–717. doi: 10.1073/pnas.71.3.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weisblum B., Davies J. Antibiotic inhibitors of the bacterial ribosome. Bacteriol Rev. 1968 Dec;32(4 Pt 2):493–528. [PMC free article] [PubMed] [Google Scholar]
- Wengler G., Wengler G. Medium hypertonicity and polyribosome structure in Hela cells. The influence of hypertonicity of the growth medium on polyribosomes in Hela cells. Eur J Biochem. 1972 May;27(1):162–173. doi: 10.1111/j.1432-1033.1972.tb01822.x. [DOI] [PubMed] [Google Scholar]
- Wu R. S., Warner J. R. Cytoplasmic synthesis of nuclear proteins. Kinetics of accumulation of radioactive proteins in various cell fractions after brief pulses. J Cell Biol. 1971 Dec;51(3):643–652. doi: 10.1083/jcb.51.3.643. [DOI] [PMC free article] [PubMed] [Google Scholar]