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For Use of the Conserved Helix-Turn-Helix Motif in DNA Binding (Escherichia Coli/Operator Recognition/Hydroxylamine Mutagenesis/Tetracycline Resistance) PAUL J

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For Use of the Conserved Helix-Turn-Helix Motif in DNA Binding (Escherichia Coli/Operator Recognition/Hydroxylamine Mutagenesis/Tetracycline Resistance) PAUL J Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6226-6230, September 1985 Genetics Dominant negative mutations in the TnIO tet repressor: Evidence for use of the conserved helix-turn-helix motif in DNA binding (Escherichia coli/operator recognition/hydroxylamine mutagenesis/tetracycline resistance) PAUL J. ISACKSON AND KEVIN P. BERTRAND Department of Microbiology and Molecular Genetics, California College of Medicine, University of California, Irvine, CA 92717 Communicated by Charles Yanofsky, May 20, 1985 ABSTRACT The Tn1O tet repressor regulates transcrip- not yet known. These observations have led several groups tion of the tetracycline-resistance determinant in transposon to propose that many sequence-specific DNA-binding pro- Tn1O. Previous DNA sequencing studies identified a region of teins use similar helix-turn-helix structures for DNA binding tet repressor (amino acids 26-47) that is homologous to the (19-21). helix-turn-helix regions of X Cro, X repressor, and catabolite We previously reported that an amino-terminal region of gene activator protein that are implicated in sequence-specific the TnJO tet repressor shows amino acid sequence homology DNA binding. Here we report the isolation of dominant tetR with the characteristic helix-turn-helix regions of Cro, X mutations that result in tet repressors deficient in tet operator repressor, and CAP (8). Here we report that mutations in binding but that retain some capacity to form dimers with, and TWJO tetR that impair repressor-operator binding, but not thereby inactivate, wild-type repressor monomers. The muta- tetracycline binding or subunit aggregation, are clustered in tions were isolated by transforming a MeMR+ tetA-lacZ fusion the region of helix-turn-helix sequence homology. strain with hydroxylamine-mutagenized leiR plasmid DNA and then screening for increased lacZ expression, DNA sequence MATERIALS analysis of 35 independent isolates identified seven different AND METHODS mutations, five of which are in the region of helix-turn-helix Bacterial Strains, Phages, and Plasmids. NK5031 (6) and sequence homology. In vitro binding studies indicate that the MO (6) are E. coli K-12 strains. XRStetl58-50 (22) is a bet' mutations in this region of let repressor reduce the affinity of gam' cIII' cI derivative of the TnlO tetA-lacZ operon let repressor for tet operator DNA by at least a factor of 1000 fusion phage XRStetl58-43 (6). NK5031(XRStetl58-50) but have no significant effect on the affinity of tet repressor for srlC300::TnlO was constructed by transduction of NK5031- tetracycline. These results provide strong support for the (ARStetl58-50) with a Plvir lysate prepared on JC10240 (23). proposal that tet repressor utilizes the conserved helix-turn- Plasmids pBT402 (8) and pBI501 have the 701-base-pair (bp) helix structural motif in binding to let operator DNA. tetR' HincIH fragment of TnWO inserted in the HincII sites of pACYC177 (24) and pUC8 (25), respectively. The tetracycline-resistance determinant in transposon TnJO Mutant Selection. pBT402 DNA (20 pug) was incubated in 1 consists of two genes, the resistance gene (tetA) and the ml of 0.8 M hydroxylamine HCl (Sigma)/50 mM sodium repressor gene (tetR), that are transcribed from divergent phosphate, pH 6/1 mM Na2EDTA for 48 hr at 370C. The overlapping promoters (1-3). Tetracycline induces transcrip- mutagenized DNA was diluted with 5 vol of water, dialyzed tion ofboth tetA and tetR by binding to tet repressor, thereby against 20 mM Tris HCl, pH 8/20mM NaCl/1 mM Na2EDTA reducing tet repressor's affinity for two operator sites that at 40C, ethanol precipitated, and then used to transform overlap the tet promoters (2-6). Both the free and operator- NK5031(Xtetl58-50) srlC300: :TnJO. Transformants were se- bound forms of tet repressor are dimers ofthe 23,300-Da tetR lected on lactose/MacConkey agar (26) containing 100 tug of polypeptide (4, 7, 8). neomycin sulfate per ml. After overnight incubation at 370C The three-dimensional structures of three sequence-spe- and 2-4 days at room temperature, pink colonies were visible cific DNA-binding proteins-the X Cro protein (9), the amongst a background of 1000-10,000 white colonies per Escherichia coli catabolite gene activator protein (CAP) plate. complex with cAMP (10), and the amino-terminal DNA- DNA Sequencing. Plasmid DNA was prepared from 2-ml binding domain ofthe XcI repressor (11)-have recently been cultures (27), incubated with RNase A (50 ,Ag/ml) for 15 min determined. Model building studies based on the protein at 370C, phenol extracted, ethanol precipitated, linearized by crystal structures, chemical-protection and chemical-modi- digestion with BamHI, phenol extracted, and ethanol pre- ficatioh data, and genetic analyses have led to detailed cipitated. One-fourth of the sample was annealed with 2.5 ng predictions about the way in which these three proteins of a tetR-specific oligonucleotide primer by heating for 5 min contact their DNA binding sites (12-19). Each of these at 950C and quickly cooling on ice. The template-primer proteins binds to operator DNA as a dimer. In the proposed mixture was then treated according to standard dideoxy- models, each protein uses pairs of a-helices (one from each sequencing procedures (28). Primer 1 (5'-CTCTACA- subunit ofthe protein) to contact successive major grooves in CCTAGC-3') corresponds to amino acids 34-38 of tet re- right-handed B-DNA. The backbone structures of these pressor; primer 2 (TGCCAGCTTTCCC) corresponds to a-helical units-two a-helices connected by a a-turn-are in amino acids 73-76; primer 3 (CATAAAAAGGCTA) corre- each case nearly identical. Moreover, there is limited but sponds to amino acids 118-121; primer 4 (AGCGACTTGAT- significant amino acid sequence homology between the GCTC) corresponds to amino acids 150-154; primer 5 helix-turn-helix regions of Cro, X repressor, and CAP and (CTAATCCGCATATGA) corresponds to amino acids regions of other DNA-binding proteins whose structures are 193-197; and primer 6 (ATCTTGGTTACCG) corresponds to the region 41-53 bp 3' to tetR. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: CAP, catabolite gene activator protein; bp, base in accordance with 18 U.S.C. §1734 solely to indicate this fact. pair(s). 6226 Downloaded by guest on September 23, 2021 Genetics: Isackson and Bertrand Proc. Natl. Acad. Sci. USA 82 (1985) 6227 fi-Galactosidase Assays. /3-Galactosidase was assayed as colonies. Under the conditions of mutagenesis that we used, described by Miller (26). Derivatives of NK5031 were grown the frequency of pink colonies (putative tetR-d/tetR+ at 370C in LB medium (26) with or without 100 Ag of heterogenotes) was 10-3 to 10-4. Only 1-10% of all neomycin sulfate per ml, as appropriate. hydroxylamine-induced tetR- mutations appear to have a Overexpression of tet Repressors and Preparation of Ex- dominant phenotype as judged by this screening procedure. tracts for in Vitro Binding Studies. The 701-bp tetR HincII DNA Sequencing and in Vivo Characterization of Mutations. fragments from pBT402 and tetR- derivatives of pBT402 Putative tetR d mutations were initially characterized by were gel-purified and ligated with HinclI-digested pUC8 dideoxy sequencing using tetR-specific primers and plasmid DNA to generate plasmids pBI501 (tetR+) and tetR- deriv- DNA prepared directly from the tetR d/tetR+ heteroge- atives of pBI501 in which tetR transcription is under the notes. Primer 2, which allows sequencing of the helix-turn- control of the pUC8 lac promoter. Plasmid-containing deriv- helix region of tetR, was used in the initial screen. Of the 46 atives of strain MO were grown to stationary phase in 50 ml mutants examined, 32 had base changes that could be of LB medium containing 100 Ag of ampicillin per ml. Cells identified by sequencing with this primer. These comprised were harvested by centrifugation, resuspended in 15 ml of 10 six different mutations in or near the helix-turn-helix region mM TrisHCl, pH 7.6/200 mM NaCI/7 mM 2-mercapto- (Table 1). Representative isolates ofthese six mutations were ethanol, and disrupted by sonication. Phenylmethylsulfonyl selected, and their tetR genes were completely sequenced to fluoride (Sigma) was added to 0.2 mM, cell debris was confirm the presence of a single base-pair change in tetR. removed by centrifugation, and tet repressor was precipitat- Mutation 38am is an amber mutation (CAG -* TAG); how- ed from the supernatant by addition of (NH4)2SO4 to 55% ever, NK5031 contains an efficient amber suppressor (supF), saturation. The pellets were resuspended in 0.5 ml of 10 mM and the tetRd character of38am reflects the properties ofthe Tris-HCl, pH 7.6/200 mM NaCl/0.1 mM Na2EDTA/7 mM suppressed protein, which is predicted to contain a tyrosine 2-mercaptoethanol, yielding cell extracts with 10-16 mg of residue at position 38 (unpublished data). total protein per ml. To further characterize the mutant repressors, isolated DNA Binding Assays. DNA fragments containing the left- plasmid DNA was transformed into both tetR- and tetR' ward TnJO tet operator (OL) were prepared from a plasmid in tetA-lacZ strains. In contrast to pBT402, none of the mutant which the rightward tet operator (OR) is deleted (3). The plasmids significantly represses B-galactosidase synthesis in 155-bp Alu I/Hae III OL fragment from this plasmid was the tetR- background (Table 1). Thus, all six tetR d muta- inserted into the Sma I site of pUC8, and the 171-bp tions in or near the helix-turn-helix region appear to dramat- EcoRI/Sal I OL fragment from the PUC8-OL plasmid was ically reduce the affinity of tet repressor for tet operator. The used for DNA binding assays.
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