Polyamine effects on DNA-directed RNA polymerases in the ciliate Tetrahymena thermophila. In vivo- and in vitro-experiments suggesting highly specific regulative interactions.
Stimulation of growth in resting cultures of the ciliated protozoan Tetrahymena thermophila stimulated putrescine formation in a manner coordinated to transcription and partly to DNA-polymerisation. The stimulation of polyamine biosynthesis involved increased formation of the precursor L-ornithine from L-arginine as well as stimulation of the regulative key enzyme ornithine decarboxylase. In order to characterize the interrelationships between polyamines and transcription, which were suggested by these in vivo-assays, in vitro-assays were performed employing isolated pure macronuclei from Tetrahymena thermophila. The results obtained were: i) The diamine putrescine and the polyamines spermidine and spermine stimulated the incorporation of [4-14C]UTP into RNA time- and concentration dependently. This stimulation was not due to changes in the ionic strength nor to substitution for divalent cations (e.g. Mg2+ or Mn2+). The di- and polyamines did not alter the Km of RNA polymerases for the substrates, e.g. UTP. ii) Purified yeast-RNA, when added to the in vitro transcription system at concentrations capable of stimulating purified ornithine decarboxylase from Tetrahymena inhibited the RNA polymerases. The inhibitory effect of RNA on the polymerases was partly antagonized by spermidine and spermine but not by putrescine whereas the residual polymerase activity was stimulated by all three bases. iii) The stimulating effects of the di- and polyamines were synergistic but not absolutely additive, suggesting different targets for their actions. iv) Stimulation of RNA polymerases by putrescine, spermidine, or spermine after inhibition of particular enzymes by alpha-amanitin allowed to distinguish the effects on the three polymerases (I, II, and III): putrescine was not specific for any of the polymerases; spermidine was most active stimulating polymerase I and spermine was most active stimulating polymerase II, less active stimulating polymerase I but strongly inhibitory to polymerase III. v) The results obtained in the previous experiments were confirmed by electrophoretic analysis of the products formed in the in vitro transcription assays which furthermore showed that the differences between the experiments with or without polyamines were most pronounced if partly denatured calf thymus DNA was present in the assay mixture. This finding and the inhibition by RNA suggest that the factor influenced most by the polyamines is the binding of the RNA polymerases to the DNA target.
Eichler
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W
Institut für Biochemie I, Universität Heidelberg.
Corr
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D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D007700:Kinetics; D011700:Putrescine / Q000096:biosynthesis / Q000494:pharmacology*; D012313:RNA / Q000032:analysis; D013095:Spermidine / Q000494:pharmacology*; D013096:Spermine / Q000494:pharmacology*; D013768:Tetrahymena / Q000187:drug effects* / Q000201:enzymology; D014158:Transcription, Genetic
D000546:Amanitins; D013096:Spermine; D012313:RNA; D012321:DNA-Directed RNA Polymerases; D013095:Spermidine; D011700:Putrescine