Summary
The regulatory region and repressor (tetR E) gene from the class E tetracycline resistance determinant previously isolated from Enterobacteriaceae have been identified and completely sequenced. The regulatory region is located between the resistance gene and the tetR gene which have opposite polarity. The tetR gene encodes a protein consisting of 211 amino acids with a calculated molecular weight of 23.6 kDa. Cloning of the tetR gene under transcriptional control of the λ P L promoter leads to overexpression of a polypeptide with an apparent molecular weight of 26 kDa. The purified protein binds sequence specifically to DNA fragments containing putative tet operators. This property is lost in the presence of tetracycline. The relationship of the tetR E sequence to four known tetracycline resistance determinants is discussed.
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Bertrand KP, Postle K, Wray LV Jr, Reznikoff WS (1983) Overlapping divergent promoters control expression of Tn10 tetracycline resistance. Gene 23:149–156
Grantham R, Gautier C, Gony M (1980) Codon frequencies in 119 individual genes confirm consistent choices of degenerate bases according to genome type. Nucleic Acids Res 8:1893–1912
Hansen D, Hillen W (1987) Tryptophan in α-helix 3 of Tet repressor forms a sequence-specific contact with tet operator in solution. J Biol Chem 262:12269–12274
Hawley DK, McClure WR (1983) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res 11:2237–2255
Hershfield V, Boyer HW, Chow C, Helinski DR (1976) Characterization of a mini-colE1 plasmid. J Bacteriol 126:447–453
Hillen W, Klock G, Kaffenberger I, Wray LV Jr, Reznikoff WS (1982) Purification of the tet repressor and tet operator from the transposon Tn10 and characterization of their interaction. J Biol Chem 257:6605–6613
Hillen W, Schollmeier K, Gatz C (1984) 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 172:185–201
Hunkapiller MW, Hewick RM, Dreyer WJ, Hood LE (1983) Highsensitivity sequencing with a gas-phase sequenator. Methods Enzymol 91:399–413
Isackson PJ, Bertrand KP (1985) 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 USA 82:6226–6230
Klock G, Hillen W (1986) Expression, purification and operator binding of the transposon Tn1721-encoded Tet repressor. J Mol Biol 189:633–641
Klock G, Unger B, Gatz C, Hillen W, Altenbuchner J, Schmid K, Schmitt R (1985) Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants. J Bacteriol 161:326–332
Marshall B, Morrissey S, Flynn P, Levy SB (1986) A new tetracycline-resistance determinant, class E, isolated from Enterobacteriaceae. Gene 50:111–117
McMurry L, Petrucci RE Jr, Levy SB (1980) Active efflux of tetracyclcline encoded by four genetically different tetracycline resistance determinants in E. coli. Proc Natl Acad Sci USA 77:3974–3977
Mendez B, Tachibana C, Levy SB (1980) Heterogeneity of tetracycline resistance determinants. Plasmid 3:99–108
Oehmichen R, Klock G, Altschmied L, Hillen W (1984) Construction of an E. coli strain overproducing the Tn10-encoded Tet repressor and its use for large scale purification. EMBO J 3:539–543
Pabo CO, Sauer RT (1984) Protein-DNA recognition. Annu Rev Biochem 53:293–321
Postle K, Nguyen TT, Bertrand KP (1984) Nucleotide sequence of the repressor gene of the Tn10 encoded tetracycline resistance determinant. Nucleic Acids Res 12:4849–4863
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Unger B, Klock G, Hillen W (1984a) Nucleotide sequence of the repressor gene of the RA1 tetracycline resistance determinant: structural and functional comparison with three related Tet repressor genes. Nucleic Acids Res 12:7693–7703
Unger B, Becker J, Hillen W (1984b) Nucleotide sequence of the gene, protein purification and characterization of the pSC101-encoded tetracycline-resistance-gene-repressor. Gene 31:103–108
Waters S, Rogowsky P, Grinsted J, Altenbuchner J, Schmitt R (1983) The tetracycline resistance determinants of RP1 and Tn1721: nucleotide sequence analysis. Nucleic Acids Res 11:6089–6105
Wissmann A, Meier I, Hillen W (1988) Saturation mutagenesis of the Tn10-encoded tet operator O1. Identification of basepairs involved in Tet repressor recognition. J Mol Biol 202:397–406
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Communicated by J. Lengeler
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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 215, 76–80 (1988). https://doi.org/10.1007/BF00331306
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DOI: https://doi.org/10.1007/BF00331306