Molecular and General Genetics MGG

, Volume 227, Issue 1, pp 106–112 | Cite as

The isolated N-terminal DNA binding domain of thec repressor of bacteriophage16-3 is functional in DNA binding in vivo and in vitro

  • Géza Dallmann
  • Ferene Marines
  • Péter Papp
  • Miklós Gaszner
  • László Orosz


The 197 amino acid c repressor of the temperateRhizobium meliloti phage16-3 still regulates theO R operator of the phage after removal of its carboxyl terminal region. When cloned in the low-copy-number plasmid pGA46, a severely truncated variant (R1-77), which retains only the first 77 amino acids of the intact protein, repressed in vivo transcription from the phage promoterP R. When theR1-77 repressor was fused toE. coli β-galactosidase, the hybrid protein boundO R operator DNA in vitro. The behavior of fusion proteins derived from a point mutant is consistent with the assignment of DNA binding specificity to the amino-terminal region. Furthermore two repressor alleles bearingts mutations that mapped in theR1-77 region (near a helix-turn-helix motif) were also temperature sensitive for regulation of theO R site, while an 18 by “in frame” deletion mutant, which mapped in the carboxyl terminal segment, regulated theO R operator in wild-type fashion. The carboxyl terminal region of the repressor is however necessary for the control of lysogenic development of16-3.

Key words

Repressor Operator binding domain Repressor-βal fusion Temperate phage Rhizobium 


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  1. Aiba H, Krakow JS (1981) Isolation and characterization of the amino and carboxyl proximal fragments of adenosine cyclic 3′,5′-phosphate receptor protein ofEscherichia coli. Biochemistry 20:4774–4780PubMedCrossRefGoogle Scholar
  2. Anderson J, Ptashne M, Harrison SC (1984) Cocrystals of the DNA binding domain of phage434 repressor and a synthetic phage434 operator. Proc Natl Acad Sci USA 81:1307–1311PubMedCrossRefGoogle Scholar
  3. An G, Friesen JD (1979) Plasmid vehicles for direct cloning ofEscherichia coli promoters. J Bacteriol 140:400–407PubMedGoogle Scholar
  4. Bolivar F, Rodriguez RL, Greene PJ, Betlach HL, Heynecker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113PubMedCrossRefGoogle Scholar
  5. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA inEscherichia coli. J Mol Biol 41:459–472PubMedCrossRefGoogle Scholar
  6. Bowen B, Steinberg J, Laemmli UK, Weintraub H (1980) The detection of DNA binding protein by protein blotting. Nucleic Acids Res 8:1–20PubMedGoogle Scholar
  7. Brent R, Ptashne M (1985) An eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43:729–736PubMedCrossRefGoogle Scholar
  8. Casabadan MJ, Martinez-Arias A, Sharpira SK, Chou J (1983) β-galactosidase gene fusion for analyzing gene expression inEscherichia coli and yeast. Methods Enzymol 100:293–308CrossRefGoogle Scholar
  9. Case ME, Giles NH (1975) Genetic evidence on the organization and action of thega-1 gene product: A protein regulating the induction of three enzymes in quinate catabolism inNeurospora crassa. Proc Natl Acad Sci USA 72:553–557PubMedCrossRefGoogle Scholar
  10. Dallmann G, Olasz F, Orosz L (1980) Virulent mutants of temperateRhizobium meliloti phage16-3: LociavirC andavirT, and increased recombination. Mol Gen Genet 178:443–446CrossRefGoogle Scholar
  11. Dallmann G, Papp P, Orosz L (1987) Related repressor specificity of unrelated phages. Nature 330:398–401CrossRefGoogle Scholar
  12. Dorgai L, Olasz F, Berényi M, Dallmann G, Pay A, Orosz L (1981) Orientation of the genetic and physical map ofRhizobium meliloti temperate phage16-3. Mol Gen Genet 182:321–25CrossRefGoogle Scholar
  13. Dorgai L, Polner G, Jónás E, Garamszegi N, Ascher Z, Pay A, Dallmann G, Orosz L (1983) The detailed physical map of the temperate phage16-3 ofRhizobium meliloti 41. Mol Gen Genet 191:430–433PubMedCrossRefGoogle Scholar
  14. Dorgai L, Olasz F, Németh K (1986) Lysogenic control of temperate phage16-3 ofRhizobium meliloti 41 is governed by two distinct regions. Mol Gen Genet 205:568–571CrossRefGoogle Scholar
  15. Kania J, Brown DT (1976) The functional repressor parts of a tetramericlac repressor-β-galactosidase chimera are organized as dimers. Proc Natl Acad Sci USA 73:3529–3533PubMedCrossRefGoogle Scholar
  16. Kiss Gy, Dobó K, Dusha I, Breznovits A, Orosz L, Vincze É, Kondorosi A (1980) Isolation and characterization of an R-prime factor inRhizobium meliloti. J Bacteriol 141:121–128PubMedGoogle Scholar
  17. Lieb M (1976) λcI mutants: Intragenic complementation and complementation with a cI promoter mutant. Mol Gen Genet 146:291–297PubMedCrossRefGoogle Scholar
  18. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  19. Mata-Gilsinger M, Ritzenthaler P (1983) Isolation of a functionalExuR-repressor-β-galactosidase hybrid protein by use of in vitro gene fusions. Gene 25:9–20PubMedCrossRefGoogle Scholar
  20. Messing J, Vieira J (1982) A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19:269–276PubMedCrossRefGoogle Scholar
  21. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 398–404Google Scholar
  22. Minton NP (1984) Improved plasmid vectors for the isolation of translationallac gene fusions. Gene 31:269–273PubMedCrossRefGoogle Scholar
  23. Müller-Hill B, Kania J (1974)Lac repressor can be fused to β-galactosidase. Nature 249:561–562PubMedCrossRefGoogle Scholar
  24. Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106PubMedCrossRefGoogle Scholar
  25. Ogata RT, Gilbert W (1978) An amino-terminal fragment oflac repressor binds specifically tolac operator. Proc Natl Acad Sci USA 75:5851–5854PubMedCrossRefGoogle Scholar
  26. Orosz L, Sváb Z, Kondorosi A, Sík T (1973) Genetic studies inRhizobiophage 16-3. I. Genes and functions on the chromosome. Mol Gen Genet 125:341–350PubMedGoogle Scholar
  27. Orosz L (1980) Methods for analysis of the C cistron of temperate phage16-3 ofRhizobium meliloti. Genetics 94:265–276PubMedGoogle Scholar
  28. Orosz L, Rostás K, Hotchkiss RD (1980) A comparison of twopoint, three-point and deletion mapping in the C cistron ofRhizobiophage 16-3, and an explanation for recombination pattern. Genetics 94:249–263PubMedGoogle Scholar
  29. Pabo CO, Sauer RT, Sturtevant JM, Ptashne M (1979) The repressor contains two domains. Proc Natl Acad Sci USA 76:1608–1612PubMedCrossRefGoogle Scholar
  30. Pfahl M, Stockter C, Gronenborn B (1974) Genetic analysis of the active sites oflac repressor. Genetics 76:669–679PubMedGoogle Scholar
  31. Platt T, Weber K, uGanem D, Miller J (1972) Translational restarts: AUG reinitiation of alac repressor fragment. Proc Natl Acad Sci USA 69:897–901PubMedCrossRefGoogle Scholar
  32. Ptashne M (1986) A genetic switch; Gene control and phage λ. Cell Press/Blackwell Scientific Publications, Cambridge, MassachussettsGoogle Scholar
  33. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedCrossRefGoogle Scholar
  34. Sauer RT, Pabo CD, Meyer BJ, Ptashne M, Backman KC (1979) Regulatory functions of the λ repressor reside in the amino terminal domain. Nature 279:396–400PubMedCrossRefGoogle Scholar
  35. Singh H, LeBowitz JH, Baldwin AS, Sharp PA (1988) Molecular cloning of an enhancer binding protein: Isolation by screening of an expression library with a recognition site DNA. Cell 52:415–423PubMedCrossRefGoogle Scholar
  36. Wilcken-Bergman B, Müller-Hill B (1982) Sequence ofgalR gene indicates a common evolutionary origin oflac andgal repressor inEscherichia coli. Proc Natl Acad Sci USA 79:2427–2431CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Géza Dallmann
    • 1
    • 2
  • Ferene Marines
    • 1
  • Péter Papp
    • 1
  • Miklós Gaszner
    • 1
  • László Orosz
    • 1
    • 2
  1. 1.Department of GeneticsAttila József UniversitySzegedHungary
  2. 2.Agricultural Biotechnology CenterInstitute of Molecular GeneticsHungary

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