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

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.     

Saturation mutagenesis of the Tn10-encoded tet operator O1. Identification of base-pairs involved in Tet repressor recognition

Saturation mutagenesis of Tn10-encoded tet operator O1 was performed by chemical synthesis of 30 sequence variants yielding all possible point mutations of an operator half side. Their effect on Tet repressor binding was scored by an in-vivo repressor titration system. Tet repressor affinities of selected operator mutants were further characterized in vitro by dissociation rate measurements. The O1 sequence spans 19 base-pairs. Out of these, all 18 palindromic base-pairs are involved in Tet repressor recognition. The central base-pair does not contribute to sequence-specific binding of Tet repressor. At position 1 a pyrimidine residue is sufficient for maximal affinity to the repressor. At positions 2, 3 and 4, each mutation reduces repressor binding at least tenfold. Mutations at positions 5, 6, 7, 8 and 9 result in less drastic reductions of Tet repressor binding. Differential effects of mutations at a given position are used to deduce the chemical functions contacted by Tet repressor. The T.A to A.T transversion at position 9 increases Tet repressor affinity slightly, while all other mutations decrease repressor binding. The increased affinity of the wild-type tet operator O2 compared to wild-type O1 results from the addition of two favorable transversions at positions +/- 9 and an unfavorable T.A to C.G transition at position -7. Deletion or palindromic doubling of the central base-pair of the O1 palindrome reveals that the wild-type spacing of both operator half sides is crucial for efficient Tet repressor binding.     

Activity reversal of Tet repressor caused by single amino acid exchanges

We explore by extensive mutagenesis regions in the sequence allowing reversal of the allosteric response of Tet repressor. The wild type requires anhydrotetracycline for induction. About 100 mutants are presented, which, in contrast, require the drug for repression. Their mutations are clustered at the interface of the DNA- and inducer-binding domains. This interface consists of a central hydrophobic region surrounded by several hydrogen bonds. While most of the mutants described here contain two to five mutations, we found five positions in this region of TetR, at which single amino acid exchanges lead to activity reversal. They may disrupt the hydrogen-bonding network bordering the domain interface. We assume that the mutations cause a repositioning of the DNA reading head with respect to the effector binding core so that the same conformational change can result in opposite activities.     

Intragenic Suppressors of Induction-Deficient TetR Mutants: Localization and Potential Mechanism of Action

Eight Tn10 Tet repressor mutants with an induction-deficient phenotype and with primary mutations located at or close to the dimer interface were mutagenized and screened for inducibility in the presence of tetracycline. The second-site suppressors with wild-type-like operator binding activity that were obtained act, except for one, at a distance, suggesting that they contribute to conformational changes in the Tet repressor. Many of these long-range suppressors occur along the dimer interface, indicating that interactions between the monomers play an important role in Tet repressor induction.     

Tn10 tet operator mutations affecting Tet repressor recognition.

The effect of single base pair alterations of the Tn10 encoded tet operator on recognition of Tet repressor was studied in vivo using a repressor titration system and in vitro by dissociation rate determinations of the respective complexes. Both methods reveal that the two operators, O1 and O2, which are in a tandem arrangement in the wild type, are recognized with a two-fold different affinity when separated. Studies on synthetic operator sequences indicate that the Tet repressor binds with higher affinity to the non-palindromic O2 wildtype than to the respective palindromic sequences. The in vivo repressor titration system links the expression of lacZ to the affinity of tet operator to Tet repressor. It was used to isolate tet operator mutations with reduced affinity to the repressor. The in vivo and in vitro obtained results with these mutants agree quantitatively and indicate, that the GC base pairs at positions 2, 6, and 8 are involved in interaction with the Tet repressor. Their importance for recognition decreases in that order. Transitions at position 7 of the tet operator show smaller effects on recognition than transversions.     

Tet repressor binding induced curvature of tet operator DNA.

Tet repressor dimer binds to two tet operator sites spaced by 30 bp in the Tn10 encoded tet regulatory DNA. The effect of repressor binding on the gel mobility of circular permutated DNA fragments containing either one or both operator sequences is reported. The EcoRI induced bending of DNA is used to compare the results with other protein binding induced structural perturbations of DNA. Tet repressor bends a DNA fragment with a single tet operator to an angle of 42 degrees +/- 7 degrees. The apparent bend angle of DNA fragments containing the tandem tet operator arrangement occupied by two Tet repressor dimers turns out to be 52 degrees +/- 9 degrees. These results are interpreted with respect to the end to end distances of the bent DNA fragments. They indicate that either the intervening tet regulatory DNA between the operators or the bound operator sequences themselves contain additional perturbations from the canonical B-DNA structure. This finding is discussed in the light of previously obtained results from CD, neutron scattering, and electrooptical studies.     

Quantitative analysis of Tn10 Tet repressor binding to a complete set of tet operator mutants.

A saturating oligonucleotide-directed mutagenesis of both tet operators in the tet regulatory sequence was performed yielding mutants with four identical base pair exchanges at equivalent positions in the four tet operator half sides. The mutants were cloned between bipolar lacZ and galK indicator genes on a multicopy plasmid allowing the quantitative analysis of their effects in vivo. In the absence of Tet repressor the mutations lead to considerably different expression levels of both genes. They are discussed with respect to the promoter consensus sequences. In particular, the -10 region of the in vivo active tetPR2 promoter is unambiguously defined by these results. In the presence of Tet repressor most of the mutants exhibit a lower affinity for that protein as determined quantitatively by their reduced expression levels. In general, tet operator recognition is most strongly affected by alterations of base pairs near the center of the palindromic sequence. The most important position is the third base pair, followed by base pairs two, four, five and six, the latter showing similar effects as base pair one. At each position, the four possible base pairs show different affinities for Tet repressor. They are discussed according to their exposure of H-bond donors and -acceptors in the major and minor grooves of the B-DNA. The results are in agreement with major groove contacts at positions two, three and five. At position four a low potential correlation of efficiencies with the H-bonding in the minor groove is found, while mutations at position six seem to influence repressor binding by other mechanisms.     

Tet repressor-tet operator contacts probed by operator DNA-modification interference studies

Contacts between tet operator DNA and Tet repressor protein are characterized by modification interference studies. The modified DNA fragments are separated into fractions with high, intermediate and low affinities for Tet repressor by polyacrylamide gel electrophoresis. Ethylation of the phosphates with N-ethylnitrosourea reveals 12 contacts of a repressor dimer to tet operator. Eight of these contacts appear to be important for Tet repressor binding, as judged by the strong interference at these positions, while four contacts are probably less important. All of the phosphate contacts are located on the same side of the B-DNA structure. The sequences of tet operators proposed to interact with the recognition alpha-helix of Tet repressor are TCTATC in three cases and CCTATC in one case. After methylation of N-7 with dimethylsulfate, strong interference is observed at the guanine residues at positions +/- 2. None of the N-7 functions of other guanine residues seems to be involved in tight contacts to Tet repressor. Tet repressor subunits form identical phosphate and guanine N-7 contacts with each half side of the two tet operators indicating twofold dyad symmetry of the complexes. Attempts to analyze the methylation interference at the adenine N-3 sites reveal different results for the operators. Modification of DNA fragments with diethylpyrocarbonate yields hypersensitive sites in the tet operators, indicating different local DNA structures. Carbethoxylation interference studies confirm the contacts at the purines found by methylation interference. All of the sequence-specific protein-DNA contacts detected in this study are centered at the inside four base-pairs in each tet operator half side. The contacts are discussed with respect to the structure of the repressor-operator complex.     

Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.

The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.     

Tryptophan in alpha-helix 3 of Tet repressor forms a sequence-specific contact with tet operator in solution

The Tn10-encoded Tet repressor contains two tryptophan residues at positions 43 and 75. The typical tryptophan fluorescence is decreased upon binding of tet operator. The Tet repressor gene was engineered to replace either or both of the Trp codons by Phe codons. The resulting single tryptophan mutants are called F43 and F75 and the double mutant F43F75. The mutant proteins were purified to homogeneity. They recognize tet operator DNA only in the absence of the inducer tetracycline, indicating an intact tertiary structure of the engineered proteins. F75 and wild-type bind tet operator with the same association constant. The association constants of F43 and F43F75 with tet operator are about 3 orders of magnitude smaller. This indicates that Trp43 is important for tet operator recognition. Trp43 fluorescence is completely quenched in the complex with tet operator DNA while Trp75 remains unaffected. Binding to nonspecific DNA leads only to a 40% decrease of Trp43 fluorescence. This is interpreted as the contribution of the changed environment while the complete quench reflects a tight sequence-specific contact of tryptophan 43 to tet operator DNA. Trp43 is solvent-exposed, while Trp75 is buried in the hydrophobic interior of the protein. These results are discussed in light of the alpha-helix turn-alpha-helix DNA binding motif deduced from homology to other repressor proteins.     

Tet repressor mutants with altered effector binding and allostery

To learn about the correlation between allostery and ligand binding of the Tet repressor (TetR) we analyzed the effect of mutations in the DNA reading head-core interface on the effector specific TetR(i2) variant. The same mutations in these subdomains can lead to completely different activities, e.g. the V99G exchange in the wild-type leads to corepression by 4-ddma-atc without altering DNA binding. However, in TetR(i2) it leads to 4-ddma-atc dependent repression in combination with reduced DNA binding in the absence of effector. The thermodynamic analysis of effector binding revealed decreased affinities and positive cooperativity. Thus, mutations in this interface can influence DNA binding as well as effector binding, albeit both ligand binding sites are not in direct contact to these altered residues. This finding represents a novel communication mode of TetR. Thus, allostery may not only operate by the structural change proposed on the basis of the crystal structures.     

The role of the N terminus in Tet repressor for tet operator binding determined by a mutational analysis.

The N-terminal residues preceding the alpha-helix-turn-alpha-helix motif on the Tn10 Tet repressor protein were probed by oligonucleotide-directed deletion mutagenesis for their role in protein activity. All deletion mutants showed decreased repression in vivo, emphasizing the importance of the N terminus for tet operator binding. Only two of the mutants, TetR delta 2-23 and TetR delta 3-8 displayed a reduced intracellular protein level. The remaining deletion mutants showed either reduced binding to tet operator and inducibility by tetracycline or transdominance. We conclude that these deletions do not affect stability and overall protein structure. DNA binding activities of residue-wise increasing deletions, TetR delta 9 through TetR delta 9-13, reveal a pattern consistent with an alpha-helical structure of the affected residues. This conclusion is supported by the helical wheel projection and the hydrophobic moment profile calculated for the protein segment ranging from residues S7-V20. We propose that these residues form an amphipathic alpha-helix which packs closely against the alpha-helix-turn-alpha-helix motif and is essential for Tet repressor activity. The residues preceding this putative alpha-helix contribute to DNA binding, but no direct interactions with base pairs of tet operator were revealed in a loss of contact analysis. Individual mutation of the 4 charged residues to alanine at the N terminus shows that no single residue can account for the reduction in repression observed for the deletion mutants.     

Tet repressor-tetracycline interaction

Previous studies [Wasylewskiet al. (1996),J. Protein Chem. 15, 45–58] have shown that the W43 residue localized within the helix-turn-helix structure domain of Tet repressor can exist in the ground state in two conformational states. In this paper we investigate the fluorescence properties of W43 of TetR upon binding of tetracycline inducer and its chemical analogs such as anhydro- and epitetracycline. Binding of the drug inducer to the protein indicates that the W43 residue still exists in two conformational states; however, its environment changes drastically, as can be judged by the changes in fluorescence parameters. The FQRS (fluorescence-quenching-resolved spectra) method was used to decompose the total emission spectrum. The resolved spectra exhibit maxima of fluorescence at 346 and 332 nm and the component quenchable by KI (346 nm) is shifted 9 nm toward the blue side of the spectrum upon inducer binding. The observed shift does not result from the changes in the exposure of W43, since the bimolecular quenching rate constant remains the same and is equal to about 2.7×10⁹M^–1sec^–1. The binding of tetracycline leads to drastic decrease of the W43 fluorescence intensity and increase of the tetracycline intensity as well as the decrease of fluorescence lifetime, especially of the W43 component characterized by the emission at 332 nm. The observed energy transfer from W43 to tetracycline is more efficient for the state characterized by the fluorescence emission at 332 nm (88%) than for the component quenchable by iodide (53%) Tetracycline and several of its derivatives were also used to observe how chemical modifications of the hydrophilic groups in tetracycline influence the mechanism of binding of the antibiotic to Tet repressor. By use of pulsed-laser photoacoustic spectroscopy it is shown that the binding of tetracyclines to Tet repressor leads to significant increase of tetracycline fluorescence quantum yields. Steady-state fluorescence quenching of tetracycline analogs in complexes with Tet repressor using potassium iodide as a quencher allowed us to determine the dependence of the exposure of bound antibiotic on the modifications of hydrophilic substituents of tetracycline. Circular dichroism studies of the TetR-[Mg · tc]⁺ complex do not indicate dramatic changes in the secondary structure of the protein; however, the observed small decrease in the TetR helicity may occur due to partial unfolding of the DNA recognition helix of the protein. The observed changes may play an important role in the process of induction in which tetracycline binding results in the loss of specific DNA binding.Abbreviations FQRS fluorescence-quenching-resolved spectra - HTH helix-turn-helix motif - tc tetracycline - TetR tetracycline repressor from Escherichia coli - TetR WT wild-type TetR - TetR W43 single point mutant with phenylalanine substituted for tryptophan at position 75 in both subunits     

A fluorescence study of Tn10-encoded Tet repressor

Steady-state fluorescence quenching and time-resolved measurements have been performed to resolve the fluorescence contributions of the two tryptophan residues, W43 and W75, in the subunit of the homodimer of the Tet repressor fromEscherichia coli. The W43 residue is localized within the helix-turn-helix structural domain, which is responsible for sequence-specific binding of the Tet repressor to thetet operator. The W75 residue is in the protein matrix near the tetracycline-binding site. The assignment of the two residues has been confirmed by use of single-tryptophan mutants carrying either W43 or W75. The FQRS (fluorescence-quenching-resolved-spectra) method has been used to decompose the total emission spectrum of the wild-type protein into spectral components. The resolved spectra have maxima of fluorescence at 349 and 324 nm for the W43 and W75 residues, respectively. The maxima of the resolved spectra are in excellent agreement with those found using single-tryptophan-containing mutants. The fluorescence decay properties of the wild type as well as of both mutants of Tet repressor have been characterized by carrying out a multitemperature study. The decays of the wild-type Tet repressor and W43-containing mutant can be described as being of double-exponential type. The W75 mutant decay can be described by a Gaussian continuous distribution centered at 5.0 nsec with a bandwidth equal to 1.34 nsec. The quenching experiments have shown the presence of two classes of W43 emission. One of the components, exposed to solvent, has a maximum of fluorescence emission at 355 nm, with the second one at about 334 nm. The red-emitting component can be characterized by bimolecular-quenching rate constant,k _q equal to 2.6×10⁹, 2.8×10⁹, and 2.0×10⁹ M^?1 sec^?1 for acrylamide, iodide, and succinimide, respectively. The bluer component is unquenchable by any of the quenchers used. The W75 residue of the Tet repressor has quenching rate constant equal to 0.85×10⁹ and 0.28 × 10⁹ M^?1 sec^?1 for acrylamide and succinimide, respectively. These values indicate that the W75 is not deeply buried within the protein matrix. Our results indicate that the Tet repressor can exist in its ground state in two distinct conformational states which differ in the microenvironment of the W43 residue.     

Binding Interaction between Tet(M) and the Ribosome: Requirements for Binding

Tet(M) protein interacts with the protein biosynthesis machinery to render this process resistant to tetracycline by a mechanism which involves release of the antibiotic from the ribosome in a reaction dependent on GTP hydrolysis. To clarify this resistance mechanism further, the interaction of Tet(M) with the ribosome has been examined by using a gel filtration assay with radioactively labelled Tet(M) protein. The presence of GTP and 5′-guanylyl imido diphosphate, but not GDP, promoted Tet(M)-ribosome complex formation. Furthermore, thiostrepton, which inhibits the activities of elongation factor G (EF-G) and EF-Tu by binding to the ribosome, blocks stable Tet(M)-ribosome complex formation. Direct competition experiments show that Tet(M) and EF-G bind to overlapping sites on the ribosome.     

A fluorescence study of Tn10-encoded Tet repressor

Steady-state fluorescence quenching and time-resolved measurements have been performed to resolve the fluorescence contributions of the two tryptophan residues, W43 and W75, in the subunit of the homodimer of the Tet repressor fromEscherichia coli. The W43 residue is localized within the helix-turn-helix structural domain, which is responsible for sequence-specific binding of the Tet repressor to thetet operator. The W75 residue is in the protein matrix near the tetracycline-binding site. The assignment of the two residues has been confirmed by use of single-tryptophan mutants carrying either W43 or W75. The FQRS (fluorescence-quenching-resolved-spectra) method has been used to decompose the total emission spectrum of the wild-type protein into spectral components. The resolved spectra have maxima of fluorescence at 349 and 324 nm for the W43 and W75 residues, respectively. The maxima of the resolved spectra are in excellent agreement with those found using single-tryptophan-containing mutants. The fluorescence decay properties of the wild type as well as of both mutants of Tet repressor have been characterized by carrying out a multitemperature study. The decays of the wild-type Tet repressor and W43-containing mutant can be described as being of double-exponential type. The W75 mutant decay can be described by a Gaussian continuous distribution centered at 5.0 nsec with a bandwidth equal to 1.34 nsec. The quenching experiments have shown the presence of two classes of W43 emission. One of the components, exposed to solvent, has a maximum of fluorescence emission at 355 nm, with the second one at about 334 nm. The red-emitting component can be characterized by bimolecular-quenching rate constant,k _q equal to 2.6×10⁹, 2.8×10⁹, and 2.0×10⁹ M^–1 sec^–1 for acrylamide, iodide, and succinimide, respectively. The bluer component is unquenchable by any of the quenchers used. The W75 residue of the Tet repressor has quenching rate constant equal to 0.85×10⁹ and 0.28 × 10⁹ M^–1 sec^–1 for acrylamide and succinimide, respectively. These values indicate that the W75 is not deeply buried within the protein matrix. Our results indicate that the Tet repressor can exist in its ground state in two distinct conformational states which differ in the microenvironment of the W43 residue.Abbreviations FQRS fluorescence-quenching-resolved spectra - HTH helix-turn-helix motif - TetR tetracycline repressor fromE. coli - WT wild-type TetR - W43 single point mutant with phenyloalanine substituted for tryptophan at position 75 in both subunits - W75 single point mutant with phenyloalanine substituted for tryptophan at position 43 in both subunits     

Single-chain Tet transregulators

We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse ‘tet-on’ phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.     

Domain motions accompanying Tet repressor induction defined by changes of interspin distances at selectively labeled sites.

To investigate internal movements in Tet repressor (TetR) during induction by tetracycline (tc) we determined the interspin distances between pairs of nitroxide spin labels attached to specific sites by electron paramagnetic resonance (EPR) spectroscopy. For this purpose, we constructed six TetR variants with engineered cysteine pairs located in regions with presumed conformational changes. These are I22C and N47C in the DNA reading head, T152C/Q175C, A161C/Q175C and R128C/D180C near the tc-binding pocket, and T202C in the dimerization surface. All TetR mutants show wild-type activities in vivo and in vitro. The binding of tc results in a considerable decrease of the distance between the nitroxide groups attached to both I22C residues in the TetR dimer and an increase of the distance between the N47C residues. These opposite effects are consistent with a twisting motion of the DNA reading heads. Changes of the spin-spin interactions between nitroxide groups attached to residues near the tc-binding pocket demonstrate that the C-terminal end of alpha-helix 9 moves away from the protein core upon DNA binding. Alterations of the dipolar interaction between nitroxide groups at T202C indicate different conformations for tc and DNA-bound repressor also in the dimerization area. These results are used to model structural changes of TetR upon induction.     

Interaction of the Bacillus subtilis phage phi 105 repressor DNA: a genetic analysis.

The Bacillus subtilis phage phi 105 repressor specifically recognizes a 14-bp operator sequence which does not exhibit 2-fold rotational symmetry. To facilitate a genetic analysis of this sequence-dependent DNA binding a B. subtilis strain was constructed in which mutations affecting the phi 105 repressor-operator interaction cause a selectable phenotype, chloramphenicol resistance. After in vivo mutagenesis, we isolated and mapped 22 different mutations in the repressor coding sequence, 15 of which are missense substitutions. These are exclusively located in the N-terminal part (positions 1-43) of the 144 residue long polypeptide. Two nonsense mutants, at positions 70 and 89, respectively, still show partial repressor activity. These data suggest that the phi 105 repressor consists of at least two independently folding structural domains, of which the N-terminal is involved in operator binding. Twelve missense mutations are clustered in a region extending from Gln-18 to Arg-37, which we propose to be the DNA-binding alpha-helix--beta-turn--alpha-helix motif, common to all lambda Cro-like repressors. The second ('recognition') helix shows significant homology with the corresponding sequence in Tn3 resolvase, and there is also a striking similarity between the phi 105 operator and the consensus sequence for a Tn3 res half-site. Based on these observations, and on the previously isolated phi 105 0c mutants, we tentatively assign some specific contacts between base pairs from the first half of a phi 105 operator site and amino acids from the repressor's 'recognition helix'.