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. 1994 Dec 6;91(25):12046–12050. doi: 10.1073/pnas.91.25.12046

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.

S Shuman 1, Y Liu 1, B Schwer 1
PMCID: PMC45373  PMID: 7991582

Abstract

Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.

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Selected References

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  1. Bakalara N., Simpson A. M., Simpson L. The Leishmania kinetoplast-mitochondrion contains terminal uridylyltransferase and RNA ligase activities. J Biol Chem. 1989 Nov 5;264(31):18679–18686. [PubMed] [Google Scholar]
  2. Barnes D. E., Johnston L. H., Kodama K., Tomkinson A. E., Lasko D. D., Lindahl T. Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6679–6683. doi: 10.1073/pnas.87.17.6679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett W. F., Paoni N. F., Keyt B. A., Botstein D., Jones A. J., Presta L., Wurm F. M., Zoller M. J. High resolution analysis of functional determinants on human tissue-type plasminogen activator. J Biol Chem. 1991 Mar 15;266(8):5191–5201. [PubMed] [Google Scholar]
  4. 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]
  5. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  6. Fausnaugh J., Shatkin A. J. Active site localization in a viral mRNA capping enzyme. J Biol Chem. 1990 May 5;265(13):7669–7672. [PubMed] [Google Scholar]
  7. Fikes J. D., Becker D. M., Winston F., Guarente L. Striking conservation of TFIID in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Nature. 1990 Jul 19;346(6281):291–294. doi: 10.1038/346291a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Gibbs C. S., Zoller M. J. Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions. J Biol Chem. 1991 May 15;266(14):8923–8931. [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Jahn D., Pande S. Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae. II. Catalytic mechanism. J Biol Chem. 1991 Dec 5;266(34):22832–22836. [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Liu J. J., McLennan A. G. Purification and properties of GTP:GTP guanylyltransferase from encysted embryos of the brine shrimp Artemia. J Biol Chem. 1994 Apr 22;269(16):11787–11794. [PubMed] [Google Scholar]
  16. Mizumoto K., Kaziro Y. Messenger RNA capping enzymes from eukaryotic cells. Prog Nucleic Acid Res Mol Biol. 1987;34:1–28. doi: 10.1016/s0079-6603(08)60491-2. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Seliger L. S., Zheng K., Shatkin A. J. Complete nucleotide sequence of reovirus L2 gene and deduced amino acid sequence of viral mRNA guanylyltransferase. J Biol Chem. 1987 Dec 5;262(34):16289–16293. [PubMed] [Google Scholar]
  22. Shabarova Z. A. Synthetic nucleotide-peptides. Prog Nucleic Acid Res Mol Biol. 1970;10:145–182. doi: 10.1016/s0079-6603(08)60564-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. 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]

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