Molecular and General Genetics MGG

, Volume 212, Issue 2, pp 287–294 | Cite as

Regulation of pyrC expression in Salmonella typhimurium: Identification of a regulatory region

  • Rod A. Kelln
  • Jan Neuhard
Article
Received:

Summary

Deletion analysis of a plasmid carrying the entire pyrC gene of Salmonella typhimurium served to localize the regulatory region within a 120 base pair DNA fragment comprising the promoter-leader region and the first 10 codons of pyrC. A region of dyad symmetry is present in the leader DNA and may result in the formation of a stable hairpin in the transcript with part of the Shine-Dalgarno sequence included in the stem. Four independently-isolated regulatory mutants, overexpressing pyrC, were found to have point mutations within the symmetry region and, significantly, the mutations occurred in sequences pertaining to either side of the stem of the putative hairpin of the transcript. All four mutations would decrease the stability of the hairpin, suggesting that pyrC expression is controlled at the level of translation. Additional evidence for translational control was provided by the finding that synthesis of galactokinase mediated from a pyrC-galK transcriptional fusion is not regulated by pyrimidines. The importance of the symmetry region in the leader was further emphasized by showing that pyrC expression is strongly affected when this region is deleted, inverted, or structured as a tandem duplication.

Key words

pyrC Dihydroorotase Translational control Regulation 
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Backstrom D, Sjoberg R-M, Lundberg LG (1986) Nucleotide sequence of the structural gene for dihydroorotase of Escherichia coli K12. Eur J Biochem 160:77–82Google Scholar
  2. Bonekamp F, Clemmesen K, Karlstrom O, Jensen KF (1984) Mechanism of UTP-modulated attenuation at the pyrE gene of Escherichia coli: an example of operon polarity control through the coupling of translation to transcription. EMBO J 3:2857–2861Google Scholar
  3. Clemmesen K, Bonekamp F, Karlstrom O, Jensen KF (1985) Role of translation in the UTP-modulated attenuation at the pyrBI operon of Escherichia coli. Mol Gen Genet 201:247–251Google Scholar
  4. Davison J, Heusterspreute M, Merchez M, Brunel F (1984) Vectors with restriction-site banks. I. pJRD158, a 3903-bp plasmid containing 28 unique cloning sites. Gene 28:311–318Google Scholar
  5. Gold L, Pribnow D, Schneider T, Shinedling S, Singer BS, Stormo G (1981) Translational initiation in procaryotes. Annu Rev Microbiol 35:365–403Google Scholar
  6. Hove-Jensen B (1985) Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli. Mol Gen Genet 201:269–276Google Scholar
  7. Jensen KF, Larsen JN, Schack L, Sivertsen A (1984) Studies on the structure and expression of Escherichia coli pyrC, pyrD, and pyrF using the cloned genes. Eur J Biochem 140:343–352Google Scholar
  8. Jensen KF, Bonekamp F, Poulsen P (1986) Attenuation at nucleotide biosynthetic genes and amino acid biosynthetic operons of Escherichia coli. Trends Biochem Sci 11:362–365Google Scholar
  9. Kelln RA, Kinaham JJ, Foltermann KF, O'Donovan GA (1975) Pyrimidine biosynthetic enzymes of Salmonella typhimurium, repressed specifically by growth in the presence of cytidine. J Bacteriol 124:764–774Google Scholar
  10. Kelln RA, Neuhard J, Stauning L (1985) Isolation and characterization of pyrimidine mutants of Salmonella typhimurium altered in expression of pyrC, pyrD, and pyrE. Can J Microbiol 31:981–987Google Scholar
  11. Levin HL, Schachman HK (1985) Regulation of aspartate transcarbamoylase synthesis in Escherichia coli: Analysis of deletion mutations in the promoeer region of the pyrBI operon. Proc Natl Acad Sci USA 82:4643–4647Google Scholar
  12. McKenney K, Shimatake H, Court D, Schmeissner U, Brady C, Rosenberg M (1981) A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. In: Chirigjian JC, Papas TS (eds) Gene amplification and analysis, vol 2. Elsevier/North-Holland, New York, pp 383–415Google Scholar
  13. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:21–78Google Scholar
  14. Michaels G, Kelln RA, Nargang FE (1987) Cloning, nucleotide sequence and expression of the pyrBI operon of Salmonella typhimurium LT2. Eur J Biochem 166:55–61Google Scholar
  15. Minton NP (1984) Improved plasmid vectors for the isolation of translational lac gene fusions. Gene 31:269–273Google Scholar
  16. Neuhard J, Kelln RA (1985) Cloning and characterization of the pyrE gene and of pyrE:: Mud1 (ApR lac) fusions from Salmonella typhimurium. Eur J Biochem 146:597–603Google Scholar
  17. Neuhard J, Kelln RA, Stauning E (1986) Cloning and structural characterization of the Salmonella typhimurium pyrC gene encoding dihydroorotase. Eur J Biochem 157:335–342Google Scholar
  18. Pierard A, Glansdorff N, Gigot D, Crabeel M, Halleux P, Thiry L (1976) Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways. J Bacteriol 127:291–301Google Scholar
  19. Poulsen P, Jensen KF (1987) Effect of UTP and GTP pools on attenuation at the pyrE gene of Escherichia coli. Mol Gen Genet 208:152–158Google Scholar
  20. Poulsen P, Bonekamp F, Jensen KF (1984) Structure of the Escherichia coli pyrE operon and control of pyrE expression by a UTP modulated intercistronic attenuation. EMBO J 3:1783–1790Google Scholar
  21. Pratt D, Tzagoloff H, Erdahl WS (1966) Conditional lethal mutants of the filamentous coliphage M13: I. Isolation, complementation, cell killing, time of cistron action. Virology 30:397–410Google Scholar
  22. Roland KL, Powell FE, Turnbough CL (1985) Role of translation and attenuation in the control of pyrBI operon expression in Escherichia coli K12. J Bacteriol 163:991–999Google Scholar
  23. Schwartz M, Neuhard J (1975) Control of expression of the pyr genes in Salmonella typhimurium: Effects of variations in uridine and cytidine nucleotide pools. J Bacteriol 121:814–822Google Scholar
  24. Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346Google Scholar
  25. Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  26. Stanssens P, Remaut E, Fiers W (1985) Alterations upstream from the Shine-Dalgarno region and their effect on bacterial gene expression. Gene 36:211–223Google Scholar
  27. Stanssens P, Remaut E, Fiers W (1986) Inefficient translation initiation causes premature transcription termination in the lacZ gene. Cell 44:711–718Google Scholar
  28. Theisen M. Kelln RA, Neuhard J (1987) Cloning and characterization of the pyrF operon of Salmonella typhimurium. Eur J Biochem 164:613–619Google Scholar
  29. Turnbough CL, Hicks KL. Donahue JP (1983) Attenuation control of pyrBI operon expression in Escherichia coli K-12. Proc Natl Acad Sci USA 80:368–372Google Scholar
  30. Turnbough CL, Kerr KH, Funderburg WR, Donahue JP, Powell FE (1987) Nucleotide sequence and characterization of the pyrF operon of Escherichia coli K-12. J Biol Chem 262:10239–10245Google Scholar
  31. Valentin-Hansen P, Albrechtsen B, Larsen JEL (1986) DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor. EMBO J 5:2015–2021Google Scholar
  32. Wertman KF, Little JW, Mount DW (1984) Rapid mutational analysis of regulatory loci in Escherichia coli K-12 using bacteriophage M13. Proc Natl Acad Sci USA 81:3801–3805Google Scholar
  33. Zabeau M, Stanley KK (1982) Enhanced expression of cro-β-galactosidase fusion proteins under the control of the PR promoter of bacteriophage λ. EMBO J 1:1217–1224Google Scholar
  34. Zuker M, Stiegler P (1981) Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res 9:133–148Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Rod A. Kelln
    • 1
  • Jan Neuhard
    • 2
  1. 1.Department of ChemistryUniversity of ReginaReginaCanada
  2. 2.University Institute of Biological Chemistry BCopenhagen KDenmark

Personalised recommendations