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. Gel-purified fragment was dominant character of these mutations is, however, very 3'-end-labeled by incubation with the large fragment ofE. coli modest. In the tetR' background, the mutant plasmids result DNA polymerase I (Boehringer Mannheim) and [a-32P]dATP in -2-fold higher levels of f3-galactosidase than seen with (>3000 Ci/mmol; 1 Ci = 37 GBq; Amersham); labeled pACYC177. fragment was separated from unincorporated [a-32P]dATP by Of the 46 putative tetRd mutants examined, 14 did not Sephadex G-50 gel filtration. DNA binding assays were have a sequence change in the helix-turn-helix region of tetR. performed by adding cell extract (up to 10 Al containing 100 The dominant phenotype of these mutants was reexamined ,g of total protein) to 32P-labeled OL fragment (=100 pg) in by transforming the tetR+ tetA-lacZ fusion strain with a final vol of 50 ,ul of 10 mM Tris-HCl, pH 7.4/10 mM isolated plasmid DNA and then assaying 8-galactosidase MgCl2/10 mM KCl/6 mM 2-mercaptoethanol/0.1 mM activity in the transformants. Only three of these mutants Na2EDTA/25 ,ug of bovine serum albumin/30 ,ug of sonicat- produced increased levels of /-galactosidase, confirming ed herring DNA. Binding reaction mixtures were incubated their tetR-d for 15 min at room temperature and electrophoresed in 5% character. Complete sequencing of the tetR polyacrylamide gels as described by Fried and Crothers (29). region in these plasmids showed that they each contained the Tetracycline Binding Assays. Cell extract (up to 50 ,Al same single base-pair change leading to an amino acid change containing 500 ,Ag of total protein) was added to [7- at residue 96 (glycine -* glutamic acid). Since we recovered 3H]tetracycline (-10 pmol; 635 mCi/mmol; Amersham) in a multiple independent isolates of each of the seven different final vol of 100,Ad of 10 mM Tris HCI, pH 8/20 mM MgCl2/200 tetR-d mutations, it seems likely that these mutations include mM NaCl/6 mM 2-mercaptoethanol/0.1 mM Na2EDTA/50 ,ug of bovine serum albumin. Binding reaction mixtures were Table 1. Sequence changes and in vivo properties of incubated for 10 min at room temperature and filtered tetR-d mutations through nitrocellulose membranes (25 mm, BA85; Schleicher ,B-Galac- & Schuell) as described by Hillen et al. (4). Num- tosidase, Amino acid ber of units RESULTS Plasmid change Codon change isolates tetR- tetR+ Isolation of Dominant tetR Mutations. The tetA-lacZ pACYC177 4900 170 operon fusion strain NK5031(XRStetl58-50) produces high pBT402 tetR- 41 24 levels of B-galactosidase under the control of the TnJO tetA 21E Gly2l - Glu GGA -* GAA 2 4500 450 promoter-operator region; and, consequently, it forms red 27I Thr27 - Ile ACC -- ATC 4 4900 340 (Lac') colonies on lactose/MacConkey agar. Introduction of 28C Arg28 Cys CGT TGT 6 4900 360 a wild-type TnJO tetR gene, either on a chromosomal copy of 38am Gln38 Tyr CAG - TAG 4 4400 510 TnJO or on a multicopy plasmid such as pBT402, represses 39L Pro39 -Leu CCT -CTT 8 4400 390 j8-galactosidase synthesis, and these tetR+ tetA-lacZ strains 40I Thr4O - Ile ACA - ATA 8 4700 370 form white (Lac-) colonies on lactose/MacConkey agar. We 96E Gly96 - Glu GGA GAA 3 5100 540 isolated dominant tetR- mutations in the (tetR-d mutations) Mutant derivatives of were examined in the tetR- tetA-lacZ strain pBT402 plasmid pBT402 by transforming the tetR+ tetA-lacZ fusion strain NK5031(XRStetl58-50) and in a tetR+ deriv- NK5031(XRStetl58-50) srlC300: :TnJO with hydroxylamine- ative of this strain containing a chromosomal copy of TnJO. ,B- mutagenized pBT402 DNA, selecting transformants on Galactosidase levels in the tetR- background indicate whether the lactose/MacConkey/neomycin agar, and screening for pink mutant repressor alone has any activity; enzyme levels in the tetR' (weakly Lac') colonies among the background of white background indicate the extent of partial dominance. Downloaded by guest on September 23, 2021 6228 Genetics: Isackson and Bertrand Proc. Natl. Acad. Sci. USA 82 (1985) most of the hydroxylamine-induced tetR-d mutations that can be isolated by our screening procedure. As expected from the mutagenic specificity of hydroxylamine (30), all of the tetR d mutations that we sequenced involve C-G -- T-A transitions. .40 / In Vitro Binding Studies. To examine the ability of tetR-d °20 / repressors to bind tet operator DNA and tetracycline, cell extracts were prepared from strains that overproduce tet 80 C D repressor. Wild-type and mutant tetR genes were subcloned into the plasmid pUC8, such that tetR is constitutively 40 transcribed from the pUC8 lac promoter. As judged by In Coomassie blue staining of NaDodSO4/polyacrylamide gels, 20 tet repressor is the most abundant protein in strains contain- ing these plasmids. None of the tetR d mutations ,,1 -, -I--I ? ,- significant- 25 50 75 100 500 25 50 75 100 500 ly alters the mobility of tet repressor in NaDodSO4/poly- Protein (u.g) acrylamide gels, nor do any of the mutations have a signifi- cant effect on the level of tet repressor in the overproducing FIG. 2. Comparison of tetracycline-binding activity in cell ex- strains (unpublished data). tracts enriched in wild-type and mutant tet repressors. Increasing A polyacrylamide gel electrophoresis binding assay (7, 29) amounts of extract were incubated with a constant amount of was used to determine relative and [3H]tetracycline and tetracycline binding was determined by a the affinities of wild-type nitrocellulose filter binding assay. (A) No repressor (pUC8; o) and tetR-d repressors for tet operator DNA (Fig. 1). Whereas wild-type repressor (pBI5OI; e); (B) mutants 21E (o) and 27I (O); (C) binding of wild-type repressor to leftward tet operator (OL) mutants 28C (o) and 39L (o); (D) mutants 401 (o) and 96E (o). DNA can be detected with 0.01 Ag oftotal protein, no binding of tetR-d repressors to OL DNA can be detected with 10 Ag of total protein. Thus, all of the tetR-d mutations examined slight reduction in tetracycline binding for 39L and 401 reflects the slightly lower proportion of these mutant repres- (21E, 271, 28C, 39L, 401, and 96E) reduce the affinity of tet sors in the cell extracts used for the binding studies. In repressor for OL by a factor of >1000. Mutation 38am was not contrast, the affinities of the 96E and 21E repressors for examined in vitro. tetracycline are reduced by factors of >100 and -20, respec- A nitrocellulose filter binding assay (4) was used to tively. determine the relative affinities of wild-type and tetR-d repressors for tetracycline (Fig. 2). Four of the tetR-d repressors (271, 38C, 39L, and 401) have essentially the same DISCUSSION affinity for tetracycline as wild-type repressor. The apparent We isolated dominant negative mutations in the TnlO tetR gene (tetRd mutations) by mutagenizing tetR+ plasmid DNA with hydroxylamine, transforming a 1 2 3 4 5 6 7 8 9 10 tetA-lacZ fusion strain A carrying a chromosomal tetR+ allele, and screening for Lac' transformants on lactose/MacConkey indicator plates. This approach is similar to those used by Sauer and coworkers (14, 15) and by Kelley and Yanofsky (31) to isolate operator- binding mutants of the XcI repressor and the E. coli trp repressor, respectively. By analogy with dominant negative W. .* mutations in the E. coli lacI gene (lacI-d mutations) (32), we assume that the dominance of tetR-d mutations reflects the ability of tetR-d and tetR+ repressor monomers to form 2 3 4 5 6 7 8 9 mixed dimers, which are themselves inactive in binding to tet B operator DNA. Only those tetR- mutations that impair repressor-operator binding without substantially affecting SW subunit aggregation are expected to have a tetR d phenotype. As with lacId mutations, the dominance oftetRd mutations is partial; the extent of dominance presumably reflects both Amm-AM the relative and absolute levels of tetRld and tetR+ subunits present in the heterogenotes, as well as the capacity of tetR-d and tetR+ subunits to form dimers. Because of the autoreg- ulatory nature of wild-type tetR (2, 3, 6), plasmid-encoded tetRd subunits may, in fact, derepress the chromosomal FIG. 1. Comparison of tet operator-binding activity in cell ex- tracts enriched in wild-type and mutant tet repressors. Arrows tetR+ gene, thereby reducing the extent ofdominance that we indicate the positions of tet repressor-operator complex (upper detect in our screening system. arrow) and unbound operator (lower arrow) in a 5% polyacrylamide We sequenced 35 independent tetR-d mutations and ob- gel. (A) Increasing amounts of extract containing wild-type repressor tained multiple isolates of7 different mutations. Five ofthese (pBI501) were incubated with a constant amount of 32P-labeled DNA 7 tetR d mutations lead to amino acid substitutions in the fragment containing the leftward TnlO tet operator. Amounts of helix-turn-helix region of tet repressor-i.e., the region of tet extract (,ug ofprotein) are as follows: lane 1, no extract; lane 2, 0.0001 repressor (amino acid residues 26-47) that shows amino acid Mg; lane 3, 0.0005 ,g; lane 4, 0.0025 ,ug; lane 5, 0.01 ug; lane 6, 0.05 sequence homology with the helix 2-helix 3 regions of X Cro Mg; lane 7, 0.25 Mug; lane 8, 1 ug; lane 9, 10 Mg; lane 10, 100 Mg. (B) and X repressor and the helix E-helix F Extracts (10 Mg of protein) containing wild-type and mutant repres- region of CAP (Fig. sors were incubated with 32P-labeled tet operator fragment as in A. 3). Four of the 5 mutations in the helix-turn-helix region, as Lane 1, no extract; lane 2, no repressor (pUC8); lane 3, wild-type well as the 2 mutations outside the helix-turn-helix region, repressor (pBIS01); lane 4, mutant 21E; lane 5, mutant 271; lane 6, were characterized by in vitro binding studies. All 6 of the mutant 28C; lane 7, mutant 39L; lane 8, mutant 401; lane 9, mutant tetRd mutations examined in vitro (21E, 27I, 28C, 39L, 40I, 96E. and 96E) reduce the affinity of tet repressor for leftward tet Downloaded by guest on September 23, 2021 Genetics: Isackson and Bertrand Proc. Natl. Acad. Sci. USA 82 (1985) 6229

helix helix

o A A *O\A * * AA AO * /*A O \ A A XCro (14-39) Phe Gly Gin Thr Lys Thr Ala Lys Asp Leu Gly Va Tyri GIn er Ala Ile Asn LAla Ile His Ala Gly A y-s o A *0 0 0 \A A A\ * 00 A A* A XcI (31-56) Leu Ser Gi Glu Ser Val Ala Asp Lys Met Gly Met E Gin Ser Gly Val Gly ALeu Phe AGly Ile AAla IleATrArGin Ile Gin Il Val e ArgA A IAr a y AA A CAP (167-192) le Thr Arg Gin Giu lie Gly Gin lie Val Gly Cys Ser ArgGuThr Val Giy Arg le Leu Lys Met Leu Glu Asp

TnlO TetR (25-50) Leu Thr Th-]r Lys Leu Ala Gin Lys Leu Gly Val Glu FGin h3 r Leu Tyr Trp His Val Lys Asn Lys Arg Ala 25 30 35 40 45 50 Possible HA-induced Ile Ile Cys Phe Thr (Tyr) Ser Ile Lys (Tyr) Ser Ile (Tyr) Tyr Ile Trp Thr mutations in TetR Leu Asp Leu Gin Val

FIG. 3. Comparison of the helix-turn-helix sequences of X Cro, X repressor, CAP, and TnJO tet repressor (TetR). The extents of a-helix 2 and a-helix 3 of Cro are indicated above the sequences. Closed and open circles above the Cro and X repressor sequences denote buried and partially buried amino acid residues, respectively (33). Residues capped by arrowheads are predicted to interact with DNA (12, 13, 18). Boxed residues indicate positions ofamino acid substitutions that impair DNA binding (Cro, A. Pakula and R. Sauer, personal communication; cI, refs. 14 and 15; CAP, ref. 16). Possible hydroxylamine-induced amino acid substitutions in tet repressor are indicated; amino acid substitutions that were obtained in this study are underlined. Tyrosine residues in parentheses indicate supF-mediated suppression of TAG codons.

operator by a factor of >1000. Whereas the 4 mutations in the mutations that we obtained. Nevertheless, this relatively helix-turn-helix region (271, 28C, 39L, and 401) have no effect small set of tetR d mutations is remarkable in the degree to on the affinity of tet repressor for tetracycline in vitro, the 2 which it focuses on the role of residues in the helix-turn-helix mutations outside the helix-turn-helix region (21E and 96E) region that, by analogy with Cro, X repressor, and CAP, are substantially reduce the affinity of tet repressor for tetracy- predicted to make DNA contacts. These results strongly cline. Taken together, the in vivo and in vitro data indicate suggest that there is both structural and functional homology that the tetR d mutations in the helix-turn-helix region between amino acid residues 26-47 of tet repressor and the specifically impair repressor binding to tet operator DNA. conserved helix-turn-helix structures ofCro, X repressor, and The amino acid sequences ofthe proposed helix-turn-helix CAP. Genetic analyses ofthe E. coli lac repressor (34) and trp region ofTnJO tet repressor, the helix 2-helix 3 regions ofCro repressor (31) likewise support the involvement of helix-turn- and X repressor, and the helix E-helix F region of CAP are helix structures in the binding of these proteins to their DNA aligned in Fig. 3. There are 5-7 amino acid residue identities recognition sites. (19-27% sequence homology) between this region of tet Tetracycline-resistance determinants in Gram-negative repressor and each of the other sequences. More importantly, bacteria have been classified on the basis of DNA-DNA the pattern of conserved residues and residue types suggests hybridization data (35), and DNA sequences have been that this region of tet repressor forms a similar helix-turn- reported for representatives of four classes: class A, helix structure (19). Thus, the positions of hydrophobic RPI/TnJ721 (36); class B, TnJO (1, 7, 37, 38); class C, residues in tet repressor (Leu-25, Leu-30, Ala-31, Leu-34, pSC101/pBR322 (39, 40); and class D, RAl (41). In partic- Val-36, Leu-41, and Val-45) correspond exactly to hydro- ular, the region of helix-turn-helix sequence homology is phobic residues in Cro, X repressor, and CAP that are known highly conserved among the four tet repressors (Fig. 4). from the crystal structures to be buried or partially buried. Whereas the overall amino acid sequence homology between Ala-31, Gly-35, and Leu-41 in tet repressor correspond to the four tet repressors varies from 44% to 63%, the sequence highly conserved residues that are important in maintaining homology within the helix-turn-helix region (residues 25-49) the characteristic bihelical structure in Cro and X repressor. varies from 72% to %%. Moreover, nearly all of the amino By analogy with Cro, X repressor, and CAP, the hydrophilic acid substitutions that have occurred in the helix-turn-helix residues at positions 27-29, 32-33, 37-40, and 42-43 would be solvent exposed and available for contacting DNA. Pro-39 region are highly conservative (e.g., Lys -+ Arg, Gln -* Glu, and Val -* Ile). With the exception of mutation 401, all of the and His-44 are the only notable exceptions to the generally TnJO tetR-d mutations that we obtained (including 21E and conserved pattern of polar and nonpolar residues in the that are conserved among the known helix-turn-helix structures. 96E) alter residues absolutely four tet repressors. Genetic analyses ofCro, X repressor, and CAP support the models for the complexes of these proteins with their DNA The nucleotide sequences of the operators in the four related tet determinants are also homologous (Fig. 5). In binding sites. In X repressor and Cro, many of the mutations that prevent operator binding are clustered in the helix particular, the sequence TATCA (positions -5 to -1) is 2-helix 3 region, and most ofthese mutations alter amino acid absolutely conserved in the left halfof all eight operators, and residues that have been proposed to contact DNA (refs. 14 and 15; A. Pakula and R. Sauer, personal communication). hel ix helix Similarly, three mutations that change the DNA-binding 25 30 35 40 45 specificity ofCAP alter a residue in helix F (Glu-181) that has Tn1O L T TR K LA!QKKL G VQP1L Y WHVK been proposed to contact DNA, both on the basis of the RAI L T T R K LA Q L G I1JQP LT:L YWH[VHK N KR genetic data (16, 17) and independent CAP-DNA modeling pSCIOI L T TR R L ME -ER:L G V -Q Q P A: L Y W H 'F K N K R studies (18). The five tetR-d mutations in the helix-turn-helix RPI LTTR K ILAs XR V Q PiAL Y W H FR N K R, region of tet repressor are in positions that correspond to in X repressor, and CAP that are exactly residues Cro, FIG. 4. Comparison of helix-turn-helix sequences in related tet we proposed to contact DNA. By analogy, speculate that repressors. Solid lines indicate highly conserved amino acid resi- most if not all of these tetR d mutations define amino acid dues; broken lines indicate amino acid residues conserved in TnJO residues in tet repressor that make direct contacts with tet and RAl versus pSC101 and RP1. Sequence references: TnIO (7), operator DNA. The narrow mutagenic specificity of RA1 (41), pSC101 (40), and RP1/TnI721 (36). Amino acids are hydroxylamine almost certainly limited the variety of tetR d identified by the single-letter code. Downloaded by guest on September 23, 2021 6230 Genetics: Isackson and Bertrand Proc. Natl. Acad. Sci. USA 82 (1985) 3. Daniels, D. W. & Bertrand, K. P. (1985) J. Mol. Biol. 184, 9 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 9 599-610. TnlO OL A C T C: T A T C A T T A T A G A G T 4. Hillen, W., Klock, G., Kaffenberger, I., Wray, L. V. & OR T C C C. T A T C A G T A T A :G A G A Reznikoff, W. S. (1982) J. Biol. Chem. 257, 6605-6613. 5. Wray, L. V., Jr., & Reznikoff, W. S. (1983) J. Bacteriol. 156, RAI OL A C T:C: T A T C A T T G A T A G: G G A 1188-1191. OR A C T CT A T C A A T G A T A ,GI G G A 6. Bertrand, K. P., Postle, K., Wray, L. V., Jr., & Reznikoff, pSC1O1 OL A G C :T: T A T C A T C G A T A A G CT W. S. (1984) J. Bacteriol. 158, 910-919. 7. Hillen, W., Gatz, C., Altschmied, L., Schollmeier, K. & OR A G T:T: T A T C A C A G Ei1 T A IA: A TT I Meier, I. (1983) J. Mol. Biol. 169, 707-721. RP1 OL A C T:T: T A T C A C IGT G A T A 'A: A C A 8. Postle, K., Nguyen, T. T. & Bertrand, K. P. (1984) Nucleic OR AA C TT A T C A G T G A T A A:, A G A Acids Res. 12, 4849-4863. 9. Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, FIG. 5. Comparison of tet operator sequences in related tetra- B. W. (1981) Nature (London) 290, 754-758. cycline-resistance determinants. Solid lines indicate highly con- 10. McKay, D. B. & Steitz, T. A. (1981) Nature (London) 290, served nucleotides; broken lines indicate nucleotides conserved in 744-749. TnlO and RA1 versus pSC101 and RP1. The circled G residues at +2 11. Pabo, C. 0. & Lewis, M. (1982) Nature (London) 298, and the symmetrically located G residues at -2 (not shown) are 443-447. protected by TnlO tet repressor from methylation by dimethyl sulfate 12. Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, (ref. 2; unpublished data). Sequence references: TnlO (1), RA1 (41), Y. & Matthews, B. W. (1982) Nature (London) 298, 718-723. pSC101/pBR322 (39), and RP1/Tnl721 (36). 13. Lewis, M., Jeffrey, A., Wang, J., Ladner, R., Ptashne, M. & Pabo, C. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, the symmetrically related sequence TGATA (positions + 1 to 435-440. 14. Hecht, M. H., Nelson, H. C. M. & Sauer, R. T. (1983) Proc. +5) is conserved in the right half of all but the pSC101 NatI. Acad. Sci. USA 80, 2676-2680. operators. Whereas TnWO tet repressor protects the guanine 15. Nelson, H. C. M., Hecht, M. H. & Sauer, R. T. (1983) Cold residue at +2 (and -2) from methylation by dimethyl sulfate, Spring Harbor Symp. Quant. Biol. 47, 441-449. it does not protect the guanine residue at +6 (and -6) (ref. 2; 16. Ebright, R. H., Cossart, P., Gicquel-Sanzey, B. & Beckwith, unpublished data). These observations tend to suggest that J. (1984) Nature (London) 311, 232-235. the predominant base-specific contacts between the tet 17. Ebright, R. H., Cossart, P., Gicquel-Sanzey, B. & Beckwith, repressors and operators are somewhat closer to the 2-fold J. (1984) Proc. Natl. Acad. Sci. USA 81, 7274-7278. axes of the operators than is apparently the case for Cro, X 18. Weber, I. T. & Steitz, T. A. (1984) Proc. Natl. Acad. Sci. repressor, and CAP (12, 13, 17, 18). USA 81, 3973-3977. 19. Pabo, C. 0. & Sauer, R. T. (1984) Annu. Rev. Biochem. 53, In the proposed operator complexes of Cro, X repressor, 293-321. and CAP, a-helix 3 (or F) of each subunit of the dimer 20. Matthews, B. W., Ohlendorf, D. H., Anderson, W. F. & protrudes from the surface of the protein and makes se- Takeda, Y. (1982) Proc. Natl. Acad. Sci. USA 79, 1428-1432. quence-specific contacts with the edges of base pairs in 21. Sauer, R. T., Yocum, R. R., Doolittle, R. F., Lewis, M. & one-half of the operator site, and a-helix 2 (or E) makes Pabo, C. 0. (1982) Nature (London) 298, 447-451. contacts with phosphates in the DNA backbone. More 22. Smith, L. D. (1985) Dissertation (University of California, specifically, the proposed operator complexes of Cro and X Irvine). repressor predict base-specific contacts between amino acids 23. Csonka, L. N. & Clarke, A. J. (1980) J. Bacteriol. 143, 1, 2, and 6 of helix 3 and nucleotides 7.5-4.5 bp from the 529-530. 24. Chang, A. C. Y. & Cohen, S. N. (1978) J. Bacteriol. 134, 2-fold axis of the operator (12, 13). Similarly, the proposed 1141-1156. CAP-operator complexes predict base-specific contacts be- 25. Vieira, J. & Messing, J. (1982) Gene 19, 259-268. tween amino acids 1, 2, and 6 of helix F and nucleotides 7-4 26. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold bp from the 2-fold axis of the operator (17, 18). By analogy, Spring Harbor Laboratory, Cold Spring Harbor, NY). Gln-38, Pro-39, and Trp-43 in tet repressor may make 27. Holmes, D. S. & Quigley, M. (1981) Anal. Biochem. 114, base-specific operator contacts. In this context, we note a 193-197. striking similarity between the consensus operator sequence 28. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. for Cro and X repressor (TATCACCGCCGGTGATA) (19) and Acad. Sci. USA 74, 5463-5467. the consensus tet operator sequence (NNNTA TCANTGA TA- 29. Fried, M. G. & Crothers, D. M. (1981) Nucleic Acids Res. 9, NNN), as well as the presence of a glutamine residue in 6505-6525. 30. Freese, E. (1971) in Chemical Mutagens, ed. Hollaender, A. position 1 of helix 3 in Cro and X repressor and in the (Plenum, New York), Vol. 1, pp. 1-56. corresponding position in tet repressor. If, as these highly 31. Kelley, R. & Yanofsky, C. (1985) Proc. Natl. Acad. Sci. USA speculative comparisons suggest, the helix 3 homolog of tet 82, 483-487. repressor makes base-specific contacts with nucleotides 32. Miller, J. H. (1978) in The Operon, eds. Miller, J. H. & 4.5-1.5 bp from the 2-fold axis ofthe operator, the two helix-3 Reznikoff, W. S. (Cold Spring Harbor Laboratory, Cold homologs of the tet repressor dimer would presumably have Spring Harbor, NY), pp. 31-88. to be oriented somewhat differently than in Cro, X repressor, 33. Ohlendorf, D. H., Anderson, W. F., Lewis, M., Pabo, C. 0. or CAP. & Matthews, B. W. (1983) J. Mol. Biol. 159, 757-769. 34. Miller, J. H. (1984) J. Mol. Biol. 180, 205-212. We thank Charles Yanofsky for comments on the manuscript; 35. Mendez, B., Tachibana, C. & Levy, S. B. (1980) Plasmid 3, Laurie Smith for help with the in vitro binding studies; Richard 99-108. Ebright for drawing our attention to the homology between the X and 36. Waters, S. H., Rogowsky, P., Grinsted, J., Altenbuchner, J. & tet operators; and Richard Kelly, Robert Sauer, and Charles Schmitt, R. (1983) Nucleic Acids Res. 11, 6089-6105. Yanofsky for communicating their results prior to publication. This 37. Hillen, W. & Schollmeier, K. (1983) Nucleic Acids Res. 11, work was supported by grants from the Beckman Postdoctoral 525-539. Scholar Program (to P.J.I.) and the National Institutes of Health 38. Nguyen, T. T., Postle, K. & Bertrand, K. P. (1983) Gene 25, (NS07068 to P.J.I.; A116735 and A100470 to K.P.B.). 83-92. 39. Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. Biol. 1. Bertrand, K. P., Postle, K., Wray, L. V., Jr., & Reznikoff, 43, 77-90. W. S. (1983) Gene 23, 149-156. 40. Unger, B., Becker, J. & Hillen, W. (1984) Gene 31, 103-108. 2. Hillen, W., Schollmeier, K. & Gatz, C. (1984) J. Mol. Biol. 41. Unger, B., Klock, G. & Hillen, W. (1984) Nucleic Acids Res. 172, 185-201. 12, 7693-7703. Downloaded by guest on September 23, 2021