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. 1989 Feb 15;258(1):165–169. doi: 10.1042/bj2580165

Studies on the inhibition by alpha-amanitin of single-step addition reactions and productive RNA synthesis catalysed by wheat-germ RNA polymerase II.

L de Mercoyrol 1, C Job 1, D Job 1
PMCID: PMC1138336  PMID: 2467661

Abstract

The rate of formation of a single phosphodiester bond with UTP substrate, U-A primer, poly[d(A-T)] template and wheat-germ RNA polymerase II is greatly depressed in the presence of alpha-amanitin. Half-maximal inhibition occurs at 0.04 microgram/ml, in close agreement with published values for inhibition of productive RNA synthesis with class II RNA polymerases from higher-plant species. However, a sizeable proportion of U-A-U synthesis is resistant to inhibition by excess alpha-amanitin. In the additional presence of ATP, i.e. under experimental conditions permitting RNA chain elongation, the synthesis of poly[r(A-U)] is arrested after the formation of the first phosphodiester bond. The results support the contention that the main enzymic process disrupted by alpha-amanitin is the translocation step of the transcription complex along the DNA template.

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Selected References

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  1. Ackerman S., Bunick D., Zandomeni R., Weinmann R. RNA polymerase II ternary transcription complexes generated in vitro. Nucleic Acids Res. 1983 Sep 10;11(17):6041–6064. doi: 10.1093/nar/11.17.6041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chandler D. W., Gralla J. Specific binding and protection of form II SV40 deoxyribonucleic acid by ribonucleic acid polymerase II from wheat germ. Biochemistry. 1980 Apr 15;19(8):1604–1612. doi: 10.1021/bi00549a012. [DOI] [PubMed] [Google Scholar]
  3. Cleland W. W. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103–138. doi: 10.1016/0076-6879(79)63008-2. [DOI] [PubMed] [Google Scholar]
  4. Cochet-Meilhac M., Chambon P. Animal DNA-dependent RNA polymerases. 11. Mechanism of the inhibition of RNA polymerases B by amatoxins. Biochim Biophys Acta. 1974 Jun 27;353(2):160–184. doi: 10.1016/0005-2787(74)90182-8. [DOI] [PubMed] [Google Scholar]
  5. Dietrich J., Teissere M., Job C., Job D. Poly(dAT) dependent trinucleotide synthesis catalysed by wheat germ RNA polymerase II. Effects of nucleotide substrates and cordycepin triphosphate. Nucleic Acids Res. 1985 Sep 11;13(17):6155–6170. doi: 10.1093/nar/13.17.6155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Grachev M. A., Hartmann G. R., Maximova T. G., Mustaev A. A., Schäffner A. R., Sieber H., Zaychikov E. F. Highly selective affinity labelling of RNA polymerase B (II) from wheat germ. FEBS Lett. 1986 May 12;200(2):287–290. doi: 10.1016/0014-5793(86)81154-1. [DOI] [PubMed] [Google Scholar]
  7. Guilfoyle T. J., Jendrisak J. J. Plant DNA-dependent RNA polymerases: subunit structures and enzymatic properties of the class II enzymes from quiescent and proliferating tissues. Biochemistry. 1978 May 16;17(10):1860–1866. doi: 10.1021/bi00603a009. [DOI] [PubMed] [Google Scholar]
  8. Jendrisak J. J., Burgess R. R. A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II. Biochemistry. 1975 Oct 21;14(21):4639–4645. doi: 10.1021/bi00692a012. [DOI] [PubMed] [Google Scholar]
  9. Jendrisak J., Guilfoyle T. J. Eukaryotic RNA polymerase: comparative subunit structures, immunological properties, and alpha-amanitin sensitivities of the class II enzymes from higher plants. Biochemistry. 1978 Apr 4;17(7):1322–1327. doi: 10.1021/bi00600a029. [DOI] [PubMed] [Google Scholar]
  10. Jendrisak J. The use of alpha-amanitin to inhibit in vivo RNA synthesis and germination in wheat embryos. J Biol Chem. 1980 Sep 25;255(18):8529–8533. [PubMed] [Google Scholar]
  11. Job C., Dietrich J., Shire D., Teissere M., Job D. Effect of low nucleotide concentrations on abortive elongation catalysed by wheat-germ RNA polymerase II. Biochem J. 1987 May 15;244(1):151–157. doi: 10.1042/bj2440151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lescure B., Williamson V., Sentenac A. Efficient and selective initiation by yeast RNA polymerase B in a dinucleotide-primed reaction. Nucleic Acids Res. 1981 Jan 10;9(1):31–45. doi: 10.1093/nar/9.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Luse D. S., Jacob G. A. Abortive initiation by RNA polymerase II in vitro at the adenovirus 2 major late promoter. J Biol Chem. 1987 Nov 5;262(31):14990–14997. [PubMed] [Google Scholar]
  14. Riva M., Schäffner A. R., Sentenac A., Hartmann G. R., Mustaev A. A., Zaychikov E. F., Grachev M. A. Active site labeling of the RNA polymerases A, B, and C from yeast. J Biol Chem. 1987 Oct 25;262(30):14377–14380. [PubMed] [Google Scholar]
  15. Sentenac A. Eukaryotic RNA polymerases. CRC Crit Rev Biochem. 1985;18(1):31–90. doi: 10.3109/10409238509082539. [DOI] [PubMed] [Google Scholar]
  16. Vaisius A. C., Wieland T. Formation of a single phosphodiester bond by RNA polymerase B from calf thymus is not inhibited by alpha-amanitin. Biochemistry. 1982 Jun 22;21(13):3097–3101. doi: 10.1021/bi00256a010. [DOI] [PubMed] [Google Scholar]
  17. Wieland T., Faulstich H. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Crit Rev Biochem. 1978 Dec;5(3):185–260. doi: 10.3109/10409237809149870. [DOI] [PubMed] [Google Scholar]
  18. von Hippel P. H., Bear D. G., Morgan W. D., McSwiggen J. A. Protein-nucleic acid interactions in transcription: a molecular analysis. Annu Rev Biochem. 1984;53:389–446. doi: 10.1146/annurev.bi.53.070184.002133. [DOI] [PubMed] [Google Scholar]

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