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

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Summary

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.

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References

  • 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 20:4774–4780

    Article  PubMed  CAS  Google Scholar 

  • 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 81:1307–1311

    Article  PubMed  CAS  Google Scholar 

  • An G, Friesen JD (1979) Plasmid vehicles for direct cloning ofEscherichia coli promoters. J Bacteriol 140:400–407

    PubMed  CAS  Google Scholar 

  • 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 2:95–113

    Article  PubMed  CAS  Google Scholar 

  • Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA inEscherichia coli. J Mol Biol 41:459–472

    Article  PubMed  CAS  Google Scholar 

  • Bowen B, Steinberg J, Laemmli UK, Weintraub H (1980) The detection of DNA binding protein by protein blotting. Nucleic Acids Res 8:1–20

    PubMed  CAS  Google Scholar 

  • Brent R, Ptashne M (1985) An eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43:729–736

    Article  PubMed  CAS  Google Scholar 

  • Casabadan MJ, Martinez-Arias A, Sharpira SK, Chou J (1983) β-galactosidase gene fusion for analyzing gene expression inEscherichia coli and yeast. Methods Enzymol 100:293–308

    Article  Google Scholar 

  • 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 72:553–557

    Article  PubMed  CAS  Google Scholar 

  • Dallmann G, Olasz F, Orosz L (1980) Virulent mutants of temperateRhizobium meliloti phage16-3: LociavirC andavirT, and increased recombination. Mol Gen Genet 178:443–446

    Article  Google Scholar 

  • Dallmann G, Papp P, Orosz L (1987) Related repressor specificity of unrelated phages. Nature 330:398–401

    Article  CAS  Google Scholar 

  • 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 182:321–25

    Article  CAS  Google Scholar 

  • 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 191:430–433

    Article  PubMed  CAS  Google Scholar 

  • 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 205:568–571

    Article  CAS  Google Scholar 

  • 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 73:3529–3533

    Article  PubMed  CAS  Google Scholar 

  • 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 141:121–128

    PubMed  CAS  Google Scholar 

  • Lieb M (1976) λcI mutants: Intragenic complementation and complementation with a cI promoter mutant. Mol Gen Genet 146:291–297

    Article  PubMed  CAS  Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Mata-Gilsinger M, Ritzenthaler P (1983) Isolation of a functionalExuR-repressor-β-galactosidase hybrid protein by use of in vitro gene fusions. Gene 25:9–20

    Article  PubMed  CAS  Google Scholar 

  • Messing J, Vieira J (1982) A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19:269–276

    Article  PubMed  CAS  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 398–404

    Google Scholar 

  • Minton NP (1984) Improved plasmid vectors for the isolation of translationallac gene fusions. Gene 31:269–273

    Article  PubMed  CAS  Google Scholar 

  • Müller-Hill B, Kania J (1974)Lac repressor can be fused to β-galactosidase. Nature 249:561–562

    Article  PubMed  Google Scholar 

  • Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106

    Article  PubMed  CAS  Google Scholar 

  • Ogata RT, Gilbert W (1978) An amino-terminal fragment oflac repressor binds specifically tolac operator. Proc Natl Acad Sci USA 75:5851–5854

    Article  PubMed  CAS  Google Scholar 

  • Orosz L, Sváb Z, Kondorosi A, Sík T (1973) Genetic studies inRhizobiophage 16-3. I. Genes and functions on the chromosome. Mol Gen Genet 125:341–350

    PubMed  CAS  Google Scholar 

  • Orosz L (1980) Methods for analysis of the C cistron of temperate phage16-3 ofRhizobium meliloti. Genetics 94:265–276

    PubMed  Google Scholar 

  • 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 94:249–263

    PubMed  Google Scholar 

  • Pabo CO, Sauer RT, Sturtevant JM, Ptashne M (1979) The repressor contains two domains. Proc Natl Acad Sci USA 76:1608–1612

    Article  PubMed  CAS  Google Scholar 

  • Pfahl M, Stockter C, Gronenborn B (1974) Genetic analysis of the active sites oflac repressor. Genetics 76:669–679

    PubMed  CAS  Google Scholar 

  • Platt T, Weber K, uGanem D, Miller J (1972) Translational restarts: AUG reinitiation of alac repressor fragment. Proc Natl Acad Sci USA 69:897–901

    Article  PubMed  CAS  Google Scholar 

  • Ptashne M (1986) A genetic switch; Gene control and phage λ. Cell Press/Blackwell Scientific Publications, Cambridge, Massachussetts

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  • Sauer RT, Pabo CD, Meyer BJ, Ptashne M, Backman KC (1979) Regulatory functions of the λ repressor reside in the amino terminal domain. Nature 279:396–400

    Article  PubMed  CAS  Google Scholar 

  • 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 52:415–423

    Article  PubMed  CAS  Google Scholar 

  • 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 79:2427–2431

    Article  Google Scholar 

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Authors and Affiliations

  1. Department of Genetics, Attila József University, Középfasor 52, 6726, Szeged, Hungary

    Géza Dallmann, Ferene Marines, Péter Papp, Miklós Gaszner & László Orosz

  2. Agricultural Biotechnology Center, Institute of Molecular Genetics, 2101 Gödöllö, PO Box 170, Hungary

    Géza Dallmann & László Orosz

Authors
  1. Géza Dallmann
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  2. Ferene Marines
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  3. Péter Papp
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  4. Miklós Gaszner
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  5. László Orosz
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Communicated by W. Arber

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Dallmann, G., Marines, F., Papp, P. et al. The isolated N-terminal DNA binding domain of thec repressor of bacteriophage16-3 is functional in DNA binding in vivo and in vitro. Molec. Gen. Genet. 227, 106–112 (1991). https://doi.org/10.1007/BF00260714

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