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. 1993 Feb;175(4):1206–1210. doi: 10.1128/jb.175.4.1206-1210.1993

Noninducible Tet repressor mutations map from the operator binding motif to the C terminus.

B Hecht 1, G Müller 1, W Hillen 1
PMCID: PMC193041  PMID: 8432715

Abstract

We have developed a new genetic selection system for Tet repressor mutations with a noninducible phenotype for tetracycline (TetRs). Extensive chemical mutagenesis of tetR yielded 93 single-site Tet repressor mutations. They map from residue 23 preceding the alpha-helix-turn-alpha-helix operator binding motif to residue 196 close to the C terminus of the repressor. Thirty-three of the mutations are clustered between residues 95 and 117, and another 27 are clustered between residues 131 to 158. Several of the mutants were characterized quantitatively in vivo for induction by tetracycline and anhydrotetracycline. While all of these are severely reduced in tetracycline-mediated induction, only some of them are affected for anhydrotetracycline-mediated induction.

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Selected References

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

  1. Altschmied L., Baumeister R., Pfleiderer K., Hillen W. A threonine to alanine exchange at position 40 of Tet repressor alters the recognition of the sixth base pair of tet operator from GC to AT. EMBO J. 1988 Dec 1;7(12):4011–4017. doi: 10.1002/j.1460-2075.1988.tb03290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baumeister R., Müller G., Hecht B., Hillen W. Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10. Proteins. 1992 Oct;14(2):168–177. doi: 10.1002/prot.340140204. [DOI] [PubMed] [Google Scholar]
  3. Beck C. F., Mutzel R., Barbé J., Müller W. A multifunctional gene (tetR) controls Tn10-encoded tetracycline resistance. J Bacteriol. 1982 May;150(2):633–642. doi: 10.1128/jb.150.2.633-642.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berens C., Altschmied L., Hillen W. The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis. J Biol Chem. 1992 Jan 25;267(3):1945–1952. [PubMed] [Google Scholar]
  5. Degenkolb J., Takahashi M., Ellestad G. A., Hillen W. Structural requirements of tetracycline-Tet repressor interaction: determination of equilibrium binding constants for tetracycline analogs with the Tet repressor. Antimicrob Agents Chemother. 1991 Aug;35(8):1591–1595. doi: 10.1128/aac.35.8.1591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hansen D., Altschmied L., Hillen W. Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes. Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline. J Biol Chem. 1987 Oct 15;262(29):14030–14035. [PubMed] [Google Scholar]
  7. Hillen W., Gatz C., Altschmied L., Schollmeier K., Meier I. Control of expression of the Tn10-encoded tetracycline resistance genes. Equilibrium and kinetic investigation of the regulatory reactions. J Mol Biol. 1983 Sep 25;169(3):707–721. doi: 10.1016/s0022-2836(83)80166-1. [DOI] [PubMed] [Google Scholar]
  8. Hillen W., Schollmeier K., Gatz C. Control of expression of the Tn10-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the tet operon regulatory region. J Mol Biol. 1984 Jan 15;172(2):185–201. doi: 10.1016/s0022-2836(84)80037-6. [DOI] [PubMed] [Google Scholar]
  9. Isackson P. J., Bertrand K. P. Dominant negative mutations in the Tn10 tet repressor: evidence for use of the conserved helix-turn-helix motif in DNA binding. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6226–6230. doi: 10.1073/pnas.82.18.6226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  11. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  12. Rasmussen B., Noller H. F., Daubresse G., Oliva B., Misulovin Z., Rothstein D. M., Ellestad G. A., Gluzman Y., Tally F. P., Chopra I. Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome. Antimicrob Agents Chemother. 1991 Nov;35(11):2306–2311. doi: 10.1128/aac.35.11.2306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sizemore C., Wissmann A., Gülland U., Hillen W. Quantitative analysis of Tn10 Tet repressor binding to a complete set of tet operator mutants. Nucleic Acids Res. 1990 May 25;18(10):2875–2880. doi: 10.1093/nar/18.10.2875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Smith L. D., Bertrand K. P. Mutations in the Tn10 tet repressor that interfere with induction. Location of the tetracycline-binding domain. J Mol Biol. 1988 Oct 20;203(4):949–959. doi: 10.1016/0022-2836(88)90120-9. [DOI] [PubMed] [Google Scholar]
  15. Su T. Z., el-Gewely M. R. A multisite-directed mutagenesis using T7 DNA polymerase: application for reconstructing a mammalian gene. Gene. 1988 Sep 15;69(1):81–89. doi: 10.1016/0378-1119(88)90380-0. [DOI] [PubMed] [Google Scholar]
  16. Takahashi M., Altschmied L., Hillen W. Kinetic and equilibrium characterization of the Tet repressor-tetracycline complex by fluorescence measurements. Evidence for divalent metal ion requirement and energy transfer. J Mol Biol. 1986 Feb 5;187(3):341–348. doi: 10.1016/0022-2836(86)90437-7. [DOI] [PubMed] [Google Scholar]
  17. Takahashi M., Degenkolb J., Hillen W. Determination of the equilibrium association constant between Tet repressor and tetracycline at limiting Mg2+ concentrations: a generally applicable method for effector-dependent high-affinity complexes. Anal Biochem. 1991 Dec;199(2):197–202. doi: 10.1016/0003-2697(91)90089-c. [DOI] [PubMed] [Google Scholar]
  18. Tovar K., Ernst A., Hillen W. Identification and nucleotide sequence of the class E tet regulatory elements and operator and inducer binding of the encoded purified Tet repressor. Mol Gen Genet. 1988 Dec;215(1):76–80. doi: 10.1007/BF00331306. [DOI] [PubMed] [Google Scholar]
  19. Wissmann A., Baumeister R., Müller G., Hecht B., Helbl V., Pfleiderer K., Hillen W. Amino acids determining operator binding specificity in the helix-turn-helix motif of Tn10 Tet repressor. EMBO J. 1991 Dec;10(13):4145–4152. doi: 10.1002/j.1460-2075.1991.tb04992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wissmann A., Wray L. V., Jr, Somaggio U., Baumeister R., Geissendörfer M., Hillen W. Selection for Tn10 tet repressor binding to tet operator in Escherichia coli: isolation of temperature-sensitive mutants and combinatorial mutagenesis in the DNA binding motif. Genetics. 1991 Jun;128(2):225–232. doi: 10.1093/genetics/128.2.225. [DOI] [PMC free article] [PubMed] [Google Scholar]

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