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. 1983;2(4):611–615. doi: 10.1002/j.1460-2075.1983.tb01471.x

Association of an RNA 5'-triphosphatase activity with RNA guanylyltransferase partially purified from rat liver nuclei.

Y Yagi, K Mizumoto, Y Kaziro
PMCID: PMC555069  PMID: 6138253

Abstract

An RNA 5'-triphosphatase activity hydrolyzing gamma-phosphate from pppN-RNA was found to be associated with mRNA guanylyltransferase partially purified from rat liver nuclei. The activity specifically removed 32P as inorganic phosphate from [gamma-32P]pppA(pA)n, but not from [beta-32P]pppA(pA)n or from [gamma-32P]ATP. Free SH group(s) were required for its activity, and the reaction was inhibited by N-ethylmaleimide. Divalent cations were not required, but were rather inhibitory for the reaction. The RNA 5'-triphosphatase activity could not be separated from the guanylyltransferase activity through successive chromatographies on Sephadex G-150, CM-Sephadex and blue dextran-Sepharose columns. Both activities remained physically associated during sedimentation in glycerol density gradients after high salt treatment. The heat stability of the RNA 5'-triphosphatase activity was almost identical with that of the guanylyltransferase activity. These results indicate that the 69000 mol. wt. protein purified from rat liver nuclei as guanylyltransferase possesses both mRNA capping and RNA 5'-triphosphatase activities.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Banerjee A. K. 5'-terminal cap structure in eucaryotic messenger ribonucleic acids. Microbiol Rev. 1980 Jun;44(2):175–205. doi: 10.1128/mr.44.2.175-205.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bickle T. A., Pirrotta V., Imber R. A simple, general procedure for purifying restriction endonucleases. Nucleic Acids Res. 1977 Aug;4(8):2561–2572. doi: 10.1093/nar/4.8.2561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  4. Gidoni D., Kahana C., Canaani D., Groner Y. Specific in vitro initiation of transcription of simian virus 40 early and late genes occurs at the various cap nucleotides including cytidine. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2174–2178. doi: 10.1073/pnas.78.4.2174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hagenbüchle O., Schibler U. Mouse beta-globin and adenovirus-2 major late transcripts are initiated at the cap site in vitro. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2283–2286. doi: 10.1073/pnas.78.4.2283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kaufmann G., Choder M., Groner Y. Synthesis of carrier-free beta-32P-nucleosides-triphosphate in almost quantitative yields. Anal Biochem. 1980 Nov 15;109(1):198–202. doi: 10.1016/0003-2697(80)90029-9. [DOI] [PubMed] [Google Scholar]
  7. Martin S. A., Paoletti E., Moss B. Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions. J Biol Chem. 1975 Dec 25;250(24):9322–9329. [PubMed] [Google Scholar]
  8. Mizumoto K., Kaziro Y., Lipmann F. Reaction mechanism of mRNA guanylyltransferase from rat liver: isolation and characterization of a guanylyl-enzyme intermediate. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1693–1697. doi: 10.1073/pnas.79.6.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mizumoto K., Lipmann F. Transmethylation and transguanylylation in 5'-RNA capping system isolated from rat liver nuclei. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4961–4965. doi: 10.1073/pnas.76.10.4961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Monroy G., Spencer E., Hurwitz J. Purification of mRNA guanylyltransferase from vaccinia virions. J Biol Chem. 1978 Jun 25;253(12):4481–4489. [PubMed] [Google Scholar]
  11. Nishikawa Y., Chambon P. Purification of mRNA guanylyltransferase from calf thymus. EMBO J. 1982;1(4):485–492. doi: 10.1002/j.1460-2075.1982.tb01195.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Shatkin A. J. Capping of eucaryotic mRNAs. Cell. 1976 Dec;9(4 Pt 2):645–653. doi: 10.1016/0092-8674(76)90128-8. [DOI] [PubMed] [Google Scholar]
  13. Shuman S. RNA capping by HeLa cell RNA guanylyltransferase. Characterization of a covalent protein-guanylate intermediate. J Biol Chem. 1982 Jun 25;257(12):7237–7245. [PubMed] [Google Scholar]
  14. Shuman S., Surks M., Furneaux H., Hurwitz J. Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme). J Biol Chem. 1980 Dec 10;255(23):11588–11598. [PubMed] [Google Scholar]
  15. Tutas D. J., Paoletti E. Purification and characterization of core-associated polynucleotide 5'-triphosphatase from Vaccinia virus. J Biol Chem. 1977 May 10;252(9):3092–3098. [PubMed] [Google Scholar]
  16. Venkatesan S., Gershowitz A., Moss B. Modification of the 5' end of mRNA. Association of RNA triphosphatase with the RNA guanylyltransferase-RNA (guanine-7-)methyltransferase complex from vaccinia virus. J Biol Chem. 1980 Feb 10;255(3):903–908. [PubMed] [Google Scholar]
  17. Venkatesan S., Gershowitz A., Moss B. Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei. J Biol Chem. 1980 Apr 10;255(7):2829–2834. [PubMed] [Google Scholar]
  18. Venkatesan S., Moss B. Donor and acceptor specificities of HeLa cell mRNA guanylyltransferase. J Biol Chem. 1980 Apr 10;255(7):2835–2842. [PubMed] [Google Scholar]
  19. Venkatesan S., Moss B. Eukaryotic mRNA capping enzyme-guanylate covalent intermediate. Proc Natl Acad Sci U S A. 1982 Jan;79(2):340–344. doi: 10.1073/pnas.79.2.340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Walseth T. F., Johnson R. A. The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP. Biochim Biophys Acta. 1979 Mar 28;562(1):11–31. doi: 10.1016/0005-2787(79)90122-9. [DOI] [PubMed] [Google Scholar]
  21. Wang D., Furuichi Y., Shatkin A. J. Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme. Mol Cell Biol. 1982 Aug;2(8):993–1001. doi: 10.1128/mcb.2.8.993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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