Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Nov;15(11):6222–6231. doi: 10.1128/mcb.15.11.6222

Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA.

P Cong 1, S Shuman 1
PMCID: PMC230874  PMID: 7565775

Abstract

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7) methyltransferase, and transcription termination factor activities. The protein is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The capping reaction entails transfer of GMP from GTP to the 5'-diphosphate end of mRNA via a covalent enzyme-(lysyl-GMP) intermediate. The active site is situated at Lys-260 of the D1 subunit within a sequence element, KxDG (motif I), that is conserved in the capping enzymes from yeasts and other DNA viruses and at the active sites of covalent adenylylation of RNA and DNA ligases. Four additional sequence motifs (II to V) are conserved in the same order and with similar spacing among the capping enzymes and several ATP-dependent ligases. The relevance of these common sequence elements to the RNA capping reaction was addressed by mutational analysis of the vaccinia virus D1 protein. Nine alanine substitution mutations were targeted to motifs II to V. Histidine-tagged versions of the mutated D1 polypeptide were coexpressed in bacteria with the D12 subunit, and the His-tagged heterodimers were purified by Ni affinity and phosphocellulose chromatography steps. Whereas each of the mutated enzymes retained triphosphatase, methyltransferase, and termination factor activities, six of nine mutant enzymes were defective in some aspect of transguanylylation. Individual mutations in motifs III, IV, and V had distinctive effects on the affinity of enzyme for GTP, the rate of covalent catalysis (EpG formation), or the transfer of GMP from enzyme to RNA. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a conserved structural basis for covalent nucleotidyl transfer.

Full Text

The Full Text of this article is available as a PDF (586.2 KB).

Selected References

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

  1. Cong P., Shuman S. Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases. J Biol Chem. 1993 Apr 5;268(10):7256–7260. [PubMed] [Google Scholar]
  2. Cong P., Shuman S. Methyltransferase and subunit association domains of vaccinia virus mRNA capping enzyme. J Biol Chem. 1992 Aug 15;267(23):16424–16429. [PubMed] [Google Scholar]
  3. Deng L., Shuman S. A role for the H4 subunit of vaccinia RNA polymerase in transcription initiation at a viral early promoter. J Biol Chem. 1994 May 13;269(19):14323–14328. [PubMed] [Google Scholar]
  4. Fresco L. D., Buratowski S. Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6624–6628. doi: 10.1073/pnas.91.14.6624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Guo P. X., Moss B. Interaction and mutual stabilization of the two subunits of vaccinia virus mRNA capping enzyme coexpressed in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4023–4027. doi: 10.1073/pnas.87.11.4023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hagler J., Luo Y., Shuman S. Factor-dependent transcription termination by vaccinia RNA polymerase. Kinetic coupling and requirement for ATP hydrolysis. J Biol Chem. 1994 Apr 1;269(13):10050–10060. [PubMed] [Google Scholar]
  7. Hagler J., Shuman S. Nascent RNA cleavage by purified ternary complexes of vaccinia RNA polymerase. J Biol Chem. 1993 Jan 25;268(3):2166–2173. [PubMed] [Google Scholar]
  8. Heaphy S., Singh M., Gait M. J. Effect of single amino acid changes in the region of the adenylylation site of T4 RNA ligase. Biochemistry. 1987 Mar 24;26(6):1688–1696. doi: 10.1021/bi00380a030. [DOI] [PubMed] [Google Scholar]
  9. Higman M. A., Bourgeois N., Niles E. G. The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity. J Biol Chem. 1992 Aug 15;267(23):16430–16437. [PubMed] [Google Scholar]
  10. Higman M. A., Christen L. A., Niles E. G. The mRNA (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli and structural and kinetic comparison to the intact capping enzyme. J Biol Chem. 1994 May 27;269(21):14974–14981. [PubMed] [Google Scholar]
  11. Kletzin A. Molecular characterisation of a DNA ligase gene of the extremely thermophilic archaeon Desulfurolobus ambivalens shows close phylogenetic relationship to eukaryotic ligases. Nucleic Acids Res. 1992 Oct 25;20(20):5389–5396. doi: 10.1093/nar/20.20.5389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kodama K., Barnes D. E., Lindahl T. In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I. Nucleic Acids Res. 1991 Nov 25;19(22):6093–6099. doi: 10.1093/nar/19.22.6093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lindahl T., Barnes D. E. Mammalian DNA ligases. Annu Rev Biochem. 1992;61:251–281. doi: 10.1146/annurev.bi.61.070192.001343. [DOI] [PubMed] [Google Scholar]
  14. Luo Y., Mao X., Deng L., Cong P., Shuman S. The D1 and D12 subunits are both essential for the transcription termination factor activity of vaccinia virus capping enzyme. J Virol. 1995 Jun;69(6):3852–3856. doi: 10.1128/jvi.69.6.3852-3856.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Luo Y., Shuman S. RNA binding properties of vaccinia virus capping enzyme. J Biol Chem. 1993 Oct 5;268(28):21253–21262. [PubMed] [Google Scholar]
  16. Mao X., Shuman S. Intrinsic RNA (guanine-7) methyltransferase activity of the vaccinia virus capping enzyme D1 subunit is stimulated by the D12 subunit. Identification of amino acid residues in the D1 protein required for subunit association and methyl group transfer. J Biol Chem. 1994 Sep 30;269(39):24472–24479. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Niles E. G., Christen L. Identification of the vaccinia virus mRNA guanyltransferase active site lysine. J Biol Chem. 1993 Nov 25;268(33):24986–24989. [PubMed] [Google Scholar]
  20. Niles E. G., Condit R. C., Caro P., Davidson K., Matusick L., Seto J. Nucleotide sequence and genetic map of the 16-kb vaccinia virus HindIII D fragment. Virology. 1986 Aug;153(1):96–112. doi: 10.1016/0042-6822(86)90011-5. [DOI] [PubMed] [Google Scholar]
  21. Niles E. G., Lee-Chen G. J., Shuman S., Moss B., Broyles S. S. Vaccinia virus gene D12L encodes the small subunit of the viral mRNA capping enzyme. Virology. 1989 Oct;172(2):513–522. doi: 10.1016/0042-6822(89)90194-3. [DOI] [PubMed] [Google Scholar]
  22. Parks R. J., Lichty B. D., Karakis C., Evans D. H. Characterization of the Shope fibroma virus DNA ligase gene. Virology. 1994 Aug 1;202(2):642–650. doi: 10.1006/viro.1994.1385. [DOI] [PubMed] [Google Scholar]
  23. Pena L., Yáez R. J., Revilla Y., Viñuela E., Salas M. L. African swine fever virus guanylyltransferase. Virology. 1993 Mar;193(1):319–328. doi: 10.1006/viro.1993.1128. [DOI] [PubMed] [Google Scholar]
  24. Schwer B., Shuman S. Mutational analysis of yeast mRNA capping enzyme. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4328–4332. doi: 10.1073/pnas.91.10.4328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shibagaki Y., Itoh N., Yamada H., Nagata S., Mizumoto K. mRNA capping enzyme. Isolation and characterization of the gene encoding mRNA guanylytransferase subunit from Saccharomyces cerevisiae. J Biol Chem. 1992 May 15;267(14):9521–9528. [PubMed] [Google Scholar]
  26. Shuman S., Broyles S. S., Moss B. Purification and characterization of a transcription termination factor from vaccinia virions. J Biol Chem. 1987 Sep 5;262(25):12372–12380. [PubMed] [Google Scholar]
  27. Shuman S. Capping enzyme in eukaryotic mRNA synthesis. Prog Nucleic Acid Res Mol Biol. 1995;50:101–129. doi: 10.1016/s0079-6603(08)60812-0. [DOI] [PubMed] [Google Scholar]
  28. Shuman S. Catalytic activity of vaccinia mRNA capping enzyme subunits coexpressed in Escherichia coli. J Biol Chem. 1990 Jul 15;265(20):11960–11966. [PubMed] [Google Scholar]
  29. Shuman S. Functional domains of vaccinia virus mRNA capping enzyme. Analysis by limited tryptic digestion. J Biol Chem. 1989 Jun 5;264(16):9690–9695. [PubMed] [Google Scholar]
  30. 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]
  31. Shuman S., Liu Y., Schwer B. Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12046–12050. doi: 10.1073/pnas.91.25.12046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shuman S., Morham S. G. Domain structure of vaccinia virus mRNA capping enzyme. Activity of the Mr 95,000 subunit expressed in Escherichia coli. J Biol Chem. 1990 Jul 15;265(20):11967–11972. [PubMed] [Google Scholar]
  33. Shuman S., Moss B. Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA. J Biol Chem. 1988 May 5;263(13):6220–6225. [PubMed] [Google Scholar]
  34. Shuman S., Ru X. M. Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis. Virology. 1995 Aug 1;211(1):73–83. doi: 10.1006/viro.1995.1380. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Skinner M. A., Moore J. B., Binns M. M., Smith G. L., Boursnell M. E. Deletion of fowlpox virus homologues of vaccinia virus genes between the 3 beta-hydroxysteroid dehydrogenase (A44L) and DNA ligase (A50R) genes. J Gen Virol. 1994 Sep;75(Pt 9):2495–2498. doi: 10.1099/0022-1317-75-9-2495. [DOI] [PubMed] [Google Scholar]
  37. Thøgersen H. C., Morris H. R., Rand K. N., Gait M. J. Location of the adenylylation site in T4 RNA ligase. Eur J Biochem. 1985 Mar 1;147(2):325–329. doi: 10.1111/j.1432-1033.1985.tb08753.x. [DOI] [PubMed] [Google Scholar]
  38. Tomkinson A. E., Totty N. F., Ginsburg M., Lindahl T. Location of the active site for enzyme-adenylate formation in DNA ligases. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):400–404. doi: 10.1073/pnas.88.2.400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Upton C., Stuart D., McFadden G. Identification and DNA sequence of the large subunit of the capping enzyme from Shope fibroma virus. Virology. 1991 Aug;183(2):773–777. doi: 10.1016/0042-6822(91)91009-6. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Wang Y. C., Burkhart W. A., Mackey Z. B., Moyer M. B., Ramos W., Husain I., Chen J., Besterman J. M., Tomkinson A. E. Mammalian DNA ligase II is highly homologous with vaccinia DNA ligase. Identification of the DNA ligase II active site for enzyme-adenylate formation. J Biol Chem. 1994 Dec 16;269(50):31923–31928. [PubMed] [Google Scholar]
  42. Wei Y. F., Robins P., Carter K., Caldecott K., Pappin D. J., Yu G. L., Wang R. P., Shell B. K., Nash R. A., Schär P. Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination. Mol Cell Biol. 1995 Jun;15(6):3206–3216. doi: 10.1128/mcb.15.6.3206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Xu Q., Teplow D., Lee T. D., Abelson J. Domain structure in yeast tRNA ligase. Biochemistry. 1990 Jul 3;29(26):6132–6138. doi: 10.1021/bi00478a004. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES