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. 1994 Mar;176(5):1475–1481. doi: 10.1128/jb.176.5.1475-1481.1994

Purification and properties of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) from Streptomyces antibioticus.

G H Jones 1
PMCID: PMC205215  PMID: 8113189

Abstract

Two forms of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) have been purified from Streptomyces antibioticus. The larger form has an M(r) of 88,000, while the M(r) of a smaller form is 47,000. Both synthetase forms are active in the formation of guanosine 5'-triphosphate, 3'-diphosphate in reaction mixtures containing methanol. Unlike the RelA protein from Escherichia coli, the synthetases from S. antibioticus do not use GDP efficiently as a substrate. Experiments using crude extracts of S. antibioticus mycelium and the 88,000-M(r) form of guanosine pentaphosphate synthetase strongly suggest that the 47,000-M(r) species is produced by proteolysis of the larger species. This conclusion is supported by the observation that antibody to either protein reacts with the other protein. Thus, the 88,000-M(r) species may be the catalytically relevant protein in vivo. Unlike the RelA protein, the 88,000-M(r) protein is not activated by ribosomes. Modest levels of guanosine pentaphosphate synthesis were observed in mycelial extracts derived from nine other actinomycetes.

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Selected References

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

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Cashel M., Lazzarini R. A., Kalbacher B. An improved method for thin-layer chromatography of nucleotide mixtures containing 32P-labelled orthophosphate. J Chromatogr. 1969 Mar 11;40(1):103–109. doi: 10.1016/s0021-9673(01)96624-5. [DOI] [PubMed] [Google Scholar]
  3. Cashel M. The control of ribonucleic acid synthesis in Escherichia coli. IV. Relevance of unusual phosphorylated compounds from amino acid-starved stringent strains. J Biol Chem. 1969 Jun 25;244(12):3133–3141. [PubMed] [Google Scholar]
  4. Choy H. A., Jones G. H. Phenoxazinone synthase from Streptomyces antibiotics: purification of the large and small enzyme forms. Arch Biochem Biophys. 1981 Oct 1;211(1):55–65. doi: 10.1016/0003-9861(81)90429-x. [DOI] [PubMed] [Google Scholar]
  5. Fehr S., Richter D. Stringent response of Bacillus stearothermophilus: evidence for the existence of two distinct guanosine 3',5'-polyphosphate synthetases. J Bacteriol. 1981 Jan;145(1):68–73. doi: 10.1128/jb.145.1.68-73.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hara A., Sy J. Guanosine 5'-triphosphate, 3'-diphosphate 5'-phosphohydrolase. Purification and substrate specificity. J Biol Chem. 1983 Feb 10;258(3):1678–1683. [PubMed] [Google Scholar]
  7. Haseltine W. A., Block R., Gilbert W., Weber K. MSI and MSII made on ribosome in idling step of protein synthesis. Nature. 1972 Aug 18;238(5364):381–384. doi: 10.1038/238381a0. [DOI] [PubMed] [Google Scholar]
  8. Haseltine W. A., Block R. Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes. Proc Natl Acad Sci U S A. 1973 May;70(5):1564–1568. doi: 10.1073/pnas.70.5.1564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hernandez V. J., Bremer H. Escherichia coli ppGpp synthetase II activity requires spoT. J Biol Chem. 1991 Mar 25;266(9):5991–5999. [PubMed] [Google Scholar]
  10. Jones G. H. Activation of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) in Streptomyces antibioticus. J Bacteriol. 1994 Mar;176(5):1482–1487. doi: 10.1128/jb.176.5.1482-1487.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jones G. H. Combined purification of actinomycin synthetase I and 3-hydroxyanthranilic acid 4-methyltransferase from Streptomyces antibioticus. J Biol Chem. 1993 Apr 5;268(10):6831–6834. [PubMed] [Google Scholar]
  12. Jones G. H. Macromolecular synthesis in Streptomyces antibioticus: in vitro systems for aminoacylation and translation from young and old cells. J Bacteriol. 1975 Oct;124(1):364–372. doi: 10.1128/jb.124.1.364-372.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kajitani M., Ishihama A. Promoter selectivity of Escherichia coli RNA polymerase. Differential stringent control of the multiple promoters from ribosomal RNA and protein operons. J Biol Chem. 1984 Feb 10;259(3):1951–1957. [PubMed] [Google Scholar]
  14. Kelly K. S., Ochi K., Jones G. H. Pleiotropic effects of a relC mutation in Streptomyces antibioticus. J Bacteriol. 1991 Apr;173(7):2297–2300. doi: 10.1128/jb.173.7.2297-2300.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Metzger S., Dror I. B., Aizenman E., Schreiber G., Toone M., Friesen J. D., Cashel M., Glaser G. The nucleotide sequence and characterization of the relA gene of Escherichia coli. J Biol Chem. 1988 Oct 25;263(30):15699–15704. [PubMed] [Google Scholar]
  17. Mukai J., Muta S., Ozumi Y. Streptomyces nucleotide 3'-pyrophosphokinase are insensitive to stringent control. J Basic Microbiol. 1989;29(10):671–674. doi: 10.1002/jobm.3620291008. [DOI] [PubMed] [Google Scholar]
  18. Ochi K. Streptomyces relC mutants with an altered ribosomal protein ST-L11 and genetic analysis of a Streptomyces griseus relC mutant. J Bacteriol. 1990 Jul;172(7):4008–4016. doi: 10.1128/jb.172.7.4008-4016.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pedersen F. S., Kjeldgaard N. O. Analysis of the relA gene product of Escherichia coli. Eur J Biochem. 1977 Jun 1;76(1):91–97. doi: 10.1111/j.1432-1033.1977.tb11573.x. [DOI] [PubMed] [Google Scholar]
  20. Pedersen F. S., Lund E., Kjeldgaard N. O. Codon specific, tRNA dependent in vitro synthesis of ppGpp and pppGpp. Nat New Biol. 1973 May 2;243(122):13–15. [PubMed] [Google Scholar]
  21. Reiness G., Yang H. L., Zubay G., Cashel M. Effects of guanosine tetraphosphate on cell-free synthesis of Escherichia coli ribosomal RNA and other gene products. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2881–2885. doi: 10.1073/pnas.72.8.2881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Richter D., Fehr S., Harder R. The guanosine 3',5'-bis(diphosphate) (ppGpp) cycle. Comparison of synthesis and degradation of guanosine 3',5'-bis(diphosphate) in various bacterial systems. Eur J Biochem. 1979 Aug 15;99(1):57–64. doi: 10.1111/j.1432-1033.1979.tb13230.x. [DOI] [PubMed] [Google Scholar]
  23. Riggs D. L., Mueller R. D., Kwan H. S., Artz S. W. Promoter domain mediates guanosine tetraphosphate activation of the histidine operon. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9333–9337. doi: 10.1073/pnas.83.24.9333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Régnier P., Thang M. N. Membrane associated proteases in E. coli. FEBS Lett. 1973 Oct 1;36(1):31–33. doi: 10.1016/0014-5793(73)80330-8. [DOI] [PubMed] [Google Scholar]
  25. Sarubbi E., Rudd K. E., Xiao H., Ikehara K., Kalman M., Cashel M. Characterization of the spoT gene of Escherichia coli. J Biol Chem. 1989 Sep 5;264(25):15074–15082. [PubMed] [Google Scholar]
  26. Schreiber G., Metzger S., Aizenman E., Roza S., Cashel M., Glaser G. Overexpression of the relA gene in Escherichia coli. J Biol Chem. 1991 Feb 25;266(6):3760–3767. [PubMed] [Google Scholar]
  27. Schreier M. H., Erni B., Staehelin T. Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors. J Mol Biol. 1977 Nov;116(4):727–753. doi: 10.1016/0022-2836(77)90268-6. [DOI] [PubMed] [Google Scholar]
  28. Strauch E., Takano E., Baylis H. A., Bibb M. J. The stringent response in Streptomyces coelicolor A3(2). Mol Microbiol. 1991 Feb;5(2):289–298. doi: 10.1111/j.1365-2958.1991.tb02109.x. [DOI] [PubMed] [Google Scholar]
  29. Sy J. A ribosome-independent, soluble stringent factor-like enzyme isolated from a Bacillus brevis. Biochemistry. 1976 Feb 10;15(3):606–609. doi: 10.1021/bi00648a024. [DOI] [PubMed] [Google Scholar]
  30. Takano E., Gramajo H. C., Strauch E., Andres N., White J., Bibb M. J. Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2). Mol Microbiol. 1992 Oct;6(19):2797–2804. doi: 10.1111/j.1365-2958.1992.tb01459.x. [DOI] [PubMed] [Google Scholar]
  31. Xiao H., Kalman M., Ikehara K., Zemel S., Glaser G., Cashel M. Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J Biol Chem. 1991 Mar 25;266(9):5980–5990. [PubMed] [Google Scholar]

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