Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1980 Jan;141(1):58–66. doi: 10.1128/jb.141.1.58-66.1980

In Vitro Synthesis of Escherichia coli Carbamoylphosphate Synthase: Evidence for Participation of the Arginine Repressor in Cumulative Repression

Willy Lissens 1,3, Raymond Cunin 1,3, Norman Kelker 4, Nicolas Glansdorff 1,3, André Piérard 2,3
PMCID: PMC293530  PMID: 6243630

Abstract

A deoxyribonucleic acid-directed in vitro system for the synthesis of Escherichia coli carbamoylphosphate synthase has been developed, and its properties have been studied. The system uses the deoxyribonucleic acid of a lambda phage carrying the car genes (λdcarAB) as template and mediates the synthesis of both subunits of the enzyme. This newly synthesized enzyme exhibits the properties of native carbamoylphosphate synthase. A study of the in vitro synthetic capacities of S-30 extracts from strains containing either a mutated or the wild-type allele of gene argR supports earlier suggestions, based on in vivo evidence, that the argR product is involved in cumulative repression of carbamoylphosphate synthase by arginine and the pyrimidines. Repression in vitro is as efficient as in vivo. In keeping with such observation it is shown that in vitro synthesis of carbamoylphosphate synthase is repressed by partially purified arginine repressor. Evidence was obtained which indicates that arginine repression of carbamoylphosphate synthase mainly operates at the level of transcription. This was based on the design of an in vitro transcription system for gene carA, the structural gene for the light subunit of carbamoylphosphate synthase. This system also allowed us to demonstrate that free arginine is the corepressor involved in carbamoylphosphate synthase repression. The present in vitro approaches, in addition to the information they have already provided, open new possibilities for further investigations on the mechanism of cumulative repression and, in particular, on the participation of pyrimidine end products in this regulatory mechanism.

Full text

PDF
58

Selected References

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

  1. Abd-el-Al A., Ingraham J. L. Control of carbamyl phosphate synthesis in Salmonella typhimurium. J Biol Chem. 1969 Aug 10;244(15):4033–4038. [PubMed] [Google Scholar]
  2. Abdelal A. T., Ingraham J. L. Carbamylphosphate synthetase from Salmonella typhimurium. Regulations, subunit composition, and function of the subunits. J Biol Chem. 1975 Jun 25;250(12):4410–4417. [PubMed] [Google Scholar]
  3. Anderson P. M., Meister A. Control of Escherichia coli carbamyl phosphate synthetase by purine and pyrimidine nucleotides. Biochemistry. 1966 Oct;5(10):3164–3169. doi: 10.1021/bi00874a013. [DOI] [PubMed] [Google Scholar]
  4. Cleary M. L., Garvin R. T., James E. Synthesis of the Escherichia coli K12 isoenzymes of ornithine transcarbamylase, performed in vitro. Mol Gen Genet. 1977 Nov 29;157(2):155–165. doi: 10.1007/BF00267393. [DOI] [PubMed] [Google Scholar]
  5. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Courtright J. B., Henning U. Malate dehydrogenase mutants in Escherichia coli K-12. J Bacteriol. 1970 Jun;102(3):722–728. doi: 10.1128/jb.102.3.722-728.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crabeel M., Charlier D., Weyens G., Feller A., Glansdorff N., Piérard A. Repressible expression of an Escherichia coli carA gene inserted into a colicinogenic factor [proceedings]. Arch Int Physiol Biochim. 1978 Oct;86(4):912–913. [PubMed] [Google Scholar]
  8. Cunin R., Boyen A., Pouwels P., Glansdorff N., Crabeel M. Parameters of gene expression in the bipolar argECBH operon of E. coli K12. The question of translational control. Mol Gen Genet. 1975 Sep 15;140(1):51–60. doi: 10.1007/BF00268988. [DOI] [PubMed] [Google Scholar]
  9. Cunin R., Kelker N., Boyen A., Yang H., Zubay G., Glansdorff N., Maas W. K. Involvement of arginine in in vitro repression of transcription of arginine genes C, B and H in Escherichia coli K 12. Biochem Biophys Res Commun. 1976 Mar 22;69(2):377–382. doi: 10.1016/0006-291x(76)90532-5. [DOI] [PubMed] [Google Scholar]
  10. Dohi M., Inouye H., Kikuchi A., Gorini L. In vitro synthesis of ornithine transcarbamylase. J Biochem. 1978 Dec;84(6):1389–1399. doi: 10.1093/oxfordjournals.jbchem.a132261. [DOI] [PubMed] [Google Scholar]
  11. Feller A., Lissens W., Glansdorff N., Piérard A. Synthesis in vitro of the catalytic subunit of Escherichia coli aspartate carbamoyltransferase [proceedings]. Arch Int Physiol Biochim. 1978 Oct;86(4):941–942. [PubMed] [Google Scholar]
  12. Gigot D., Crabeel M., Feller A., Lissens W., Piérard A., Glansdorff N. Transcription polarity of the carAB gene cluster of Escherichia coli [proceedings]. Arch Int Physiol Biochim. 1978 Oct;86(4):915–916. [PubMed] [Google Scholar]
  13. Glansdorff N., Dambly C., Palchaudhuri S., Crabeel M., Piérard A., Halleux P. Isolation and heteroduplex mapping of a lambda transducing bacteriophage carrying the structural genes for carbamoylphosphate synthase: regulation of enzyme synthesis in Escherichia coli K-12 lysogens. J Bacteriol. 1976 Jul;127(1):302–308. doi: 10.1128/jb.127.1.302-308.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Halleux P., Crabeel M., Glansdorff N., Piérard A. Proceedings: Synthesis in vitro of Escherichia coli carbamoylphosphate synthetase. Arch Int Physiol Biochim. 1975 Feb;83(1):186–187. [PubMed] [Google Scholar]
  15. Justesen J., Neuhard J. pyrR identical to pyrH in Salmonella typhimurium: control of expression of the pyr genes. J Bacteriol. 1975 Sep;123(3):851–854. doi: 10.1128/jb.123.3.851-854.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kelker N. E., Maas W. K., Yang H. L., Zubay G. In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor. Mol Gen Genet. 1976 Feb 27;144(1):17–20. doi: 10.1007/BF00277298. [DOI] [PubMed] [Google Scholar]
  17. Kelln R. A., Kinahan J. J., Foltermann K. F., O'Donovan G. A. Pyrimidine biosynthetic enzymes of Salmonella typhimurium, repressed specifically by growth in the presence of cytidine. J Bacteriol. 1975 Nov;124(2):764–774. doi: 10.1128/jb.124.2.764-774.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kennell D., Riezman H. Transcription and translation initiation frequencies of the Escherichia coli lac operon. J Mol Biol. 1977 Jul;114(1):1–21. doi: 10.1016/0022-2836(77)90279-0. [DOI] [PubMed] [Google Scholar]
  19. Lissens W., Cunin R., Glansdorff N., Piérard A. Evidence in vitro for a participation of the arginine repressor in cumulative repression of Escherichia coli carbamoylphosphate synthase [proceedings]. Arch Int Physiol Biochim. 1979 Aug;87(3):630–632. [PubMed] [Google Scholar]
  20. Mergeay M., Gigot D., Beckmann J., Glansdorff N., Piérard A. Physiology and genetics of carbamoylphosphate synthesis in Escherichia coli K12. Mol Gen Genet. 1974;133(4):299–316. doi: 10.1007/BF00332706. [DOI] [PubMed] [Google Scholar]
  21. O'Donovan G. A., Gerhart J. C. Isolation and partial characterization of regulatory mutants of the pyrimidine pathway in Salmonella typhimurium. J Bacteriol. 1972 Mar;109(3):1085–1096. doi: 10.1128/jb.109.3.1085-1096.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Piérard A. Control of the activity of Escherichia coli carbamoyl phosphate synthetase by antagonistic allosteric effectors. Science. 1966 Dec 23;154(3756):1572–1573. doi: 10.1126/science.154.3756.1572. [DOI] [PubMed] [Google Scholar]
  23. Piérard A., Glansdorff N., Gigot D., Crabeel M., Halleux P., Thiry L. Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways. J Bacteriol. 1976 Jul;127(1):291–301. doi: 10.1128/jb.127.1.291-301.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Piérard A., Glansdorff N., Mergeay M., Wiame J. M. Control of the biosynthesis of carbamoyl phosphate in Escherichia coli. J Mol Biol. 1965 Nov;14(1):23–36. doi: 10.1016/s0022-2836(65)80226-1. [DOI] [PubMed] [Google Scholar]
  25. Piérard A., Glansdorff N., Yashphe J. Mutations affecting uridine monophosphate pyrophosphorylase or the argR gene in Escherichia coli. Effects on carbamoyl phosphate and pyrimidine biosynthesis and on uracil uptake. Mol Gen Genet. 1972;118(3):235–245. doi: 10.1007/BF00333460. [DOI] [PubMed] [Google Scholar]
  26. Piérard A., Lissens W., Halleux P., Cunin R., Glansdorff N. Role of transcriptional regulation and enzyme inactivation in the synthesis of Escherichia coli carbamoylphosphate synthase. J Bacteriol. 1980 Jan;141(1):382–385. doi: 10.1128/jb.141.1.382-385.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Piérard A., Wiame J. M. Regulation and mutation affecting a glutamine dependent formation of carbamyl phosphate in Escherichia coli. Biochem Biophys Res Commun. 1964 Feb 18;15(1):76–81. doi: 10.1016/0006-291x(64)90106-8. [DOI] [PubMed] [Google Scholar]
  28. Schwartz M., Neuhard J. Control of expression of the pyr genes in Salmonella typhimurium: effects of variations in uridine and cytidine nucleotide pools. J Bacteriol. 1975 Mar;121(3):814–822. doi: 10.1128/jb.121.3.814-822.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Urm E., Yang H., Zubay G., Kelker N., Maas W. In vitro repression of n- -acetyl-L-ornithinase synthesis in Escherichia coli. Mol Gen Genet. 1973;121(1):1–7. doi: 10.1007/BF00353688. [DOI] [PubMed] [Google Scholar]
  30. Williams J. C., O'Donovan G. A. Repression of enzyme synthesis of the pyrimidine pathway in Salmonella typhimurium. J Bacteriol. 1973 Sep;115(3):1071–1076. doi: 10.1128/jb.115.3.1071-1076.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zubay G. In vitro synthesis of protein in microbial systems. Annu Rev Genet. 1973;7:267–287. doi: 10.1146/annurev.ge.07.120173.001411. [DOI] [PubMed] [Google Scholar]
  32. Zubay G., Morse D. E., Schrenk W. J., Miller J. H. Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli. Proc Natl Acad Sci U S A. 1972 May;69(5):1100–1103. doi: 10.1073/pnas.69.5.1100. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES