Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1982 Jan;79(2):340–344. doi: 10.1073/pnas.79.2.340

Eukaryotic mRNA capping enzyme-guanylate covalent intermediate.

S Venkatesan, B Moss
PMCID: PMC345722  PMID: 6281766

Abstract

Incubation of HeLa cell mRNA guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) with [alpha-32P]GTP and a divalent cation in the absence of an RNA acceptor results in the formation of a covalent enzyme-guanylate complex. The complex, after purification by phosphocellulose chromatography, can transfer its bound GMP moiety to pyrophosphate, regenerating GTP, or to the 5'-diphosphate end of poly(A), forming a cap structure G(5')pppA(pA)n. The GMP-polypeptide has a molecular weight of 65,000 and is stable to heating in the presence of sodium dodecyl sulfate. On the basis of the alkali-stable and acid-labile nature of the bond and its susceptibility to nucleophilic attack by hydroxylamine at low pH, the GMP-polypeptide linkage appears to be a phosphoamine bond. After digestion with trypsin, a single GMP-peptide was resolved by two dimensional electrophoresis and chromatography.

Full text

PDF
340

Images in this article

341Image
on p.341
342Image
on p.342
342Image
on p.342
343Image
on p.343
344Image
on p.344

Selected References

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

  1. Ambros V., Baltimore D. Protein is linked to the 5' end of poliovirus RNA by a phosphodiester linkage to tyrosine. J Biol Chem. 1978 Aug 10;253(15):5263–5266. [PubMed] [Google Scholar]
  2. Babich A., Nevins J. R., Darnell J. E., Jr Early capping of transcripts from the adenovirus major late transcription unit. Nature. 1980 Sep 18;287(5779):246–248. doi: 10.1038/287246a0. [DOI] [PubMed] [Google Scholar]
  3. Bajszár G., Szabó G., Simoncsits A., Molnár J. Methylated cap formation by enzymes bound to nuclear informofer particles. Mol Biol Rep. 1978 Jun 16;4(2):93–96. doi: 10.1007/BF00775967. [DOI] [PubMed] [Google Scholar]
  4. Champoux J. J. DNA is linked to the rat liver DNA nicking-closing enzyme by a phosphodiester bond to tyrosine. J Biol Chem. 1981 May 25;256(10):4805–4809. [PubMed] [Google Scholar]
  5. Desiderio S. V., Kelly T. J., Jr Structure of the linkage between adenovirus DNA and the 55,000 molecular weight terminal protein. J Mol Biol. 1981 Jan 15;145(2):319–337. doi: 10.1016/0022-2836(81)90208-4. [DOI] [PubMed] [Google Scholar]
  6. Ensinger M. J., Moss B. Modification of the 5' terminus of mRNA by an RNA (guanine-7-)-methyltransferase from HeLa cells. J Biol Chem. 1976 Sep 10;251(17):5283–5291. [PubMed] [Google Scholar]
  7. Furuichi Y. "Pretranscriptional capping" in the biosynthesis of cytoplasmic polyhedrosis virus mRNA. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1086–1090. doi: 10.1073/pnas.75.3.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gumport R. I., Lehman I. R. Structure of the DNA ligase-adenylate intermediate: lysine (epsilon-amino)-linked adenosine monophosphoramidate. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2559–2563. doi: 10.1073/pnas.68.10.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hermoso J. M., Salas M. Protein p3 is linked to the DNA of phage phi 29 through a phosphoester bond between serine and 5'-dAMP. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6425–6428. doi: 10.1073/pnas.77.11.6425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Keith J. M., Ensinger M. J., Mose B. HeLa cell RNA (2'-O-methyladenosine-N6-)-methyltransferase specific for the capped 5'-end of messenger RNA. J Biol Chem. 1978 Jul 25;253(14):5033–5039. [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Langberg S. R., Moss B. Post-transcriptional modifications of mRNA. Purification and characterization of cap I and cap II RNA (nucleoside-2'-)-methyltransferases from HeLa cells. J Biol Chem. 1981 Oct 10;256(19):10054–10060. [PubMed] [Google Scholar]
  13. Martin S. A., Moss B. Modification of RNA by mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions. J Biol Chem. 1975 Dec 25;250(24):9330–9335. [PubMed] [Google Scholar]
  14. Martin S. A., Moss B. mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites. J Biol Chem. 1976 Dec 10;251(23):7313–7321. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Rothberg P. G., Harris T. J., Nomoto A., Wimmer E. O4-(5'-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4868–4872. doi: 10.1073/pnas.75.10.4868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Salditt-Georgieff M., Harpold M., Chen-Kiang S., Darnell J. E., Jr The addition of 5' cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped. Cell. 1980 Jan;19(1):69–78. doi: 10.1016/0092-8674(80)90389-x. [DOI] [PubMed] [Google Scholar]
  19. Shuman S., Hurwitz J. Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme--guanylate intermediate. Proc Natl Acad Sci U S A. 1981 Jan;78(1):187–191. doi: 10.1073/pnas.78.1.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Tse Y. C., Kirkegaard K., Wang J. C. Covalent bonds between protein and DNA. Formation of phosphotyrosine linkage between certain DNA topoisomerases and DNA. J Biol Chem. 1980 Jun 25;255(12):5560–5565. [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Wei C., Moss B. 5'-Terminal capping of RNA by guanylyltransferase from HeLa cell nuclei. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3758–3761. doi: 10.1073/pnas.74.9.3758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Weil P. A., Luse D. S., Segall J., Roeder R. G. Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA. Cell. 1979 Oct;18(2):469–484. doi: 10.1016/0092-8674(79)90065-5. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES