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.     

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.     

A possible tertiary structure change induced by acrylamide in the DNA-binding domain of the Tn10-encoded Tet repressor. A fluorescence study

A thorough investigation of the acrylamide fluorescence quenching of F75TetR, a mutant of the Tn10-encoded TetR repressor containing a single Trp residue at position 43, was carried out. The Trp-43 residue is located in a helix-turn-helix (H-t-H) motif involved in the specific binding of F75TetR to the operator site in specific DNA. Distinct Ranges of acrylamide concentration have been assumed. At acrylamide concentrations below 0.15–0.2 M (a usual range of values in fluorescence quenching studies) the observed limited tertiary structure change induced by acrylamide is consistent with a noncooperative local unfolding of the DNA-binding domain. It is suggested that penetration of the neutral quencher could cause the deletion of a hydrophobic tertiary structure contact, partly involving TrP-43, responsible for the anchoring of the H-t-H motif inside the three-helix protein bundle, characterizing the N-terminal part. Correspondingly, the affinity of the mutant repressor for the operator was shown to decrease substantially (about five orders of magnitude), seemingly losing its specificity. A subsequent phase, up to 0.8 M acrylamide, was observed in which the involved intermediate protein structure is not further perturbed, nor is DNA binding.Abbreviations Tris tris(hydroxymethyl)aminomethane - DTT dithiothreitol - FVSTetR engineered tetracycline repressor in which the Trp residue at the position 75 in the wild-type repressor TetR is replaced by a Phe residue - H-t-H helix-turn-helix super-secondary structure     

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.     

Amino acids determining operator binding specificity in the helix-turn-helix motif of Tn10 Tet repressor.

Each of 22 amino acids in the proposed alpha-helix-turn-alpha-helix operator binding motif of the Tn10 encoded Tet repressor was replaced by alanine and one residue was replaced by valine to determine their role in tet operator recognition by a 'loss of contact' analysis with 16 operator variants. One class of amino acids consisting of T27 and R28 in the first alpha-helix and L41, Y42, W43 and H44 in the recognition alpha-helix are quantitatively most important for wild-type operator binding. These residues are probably involved in the structural architecture of the motif. A second class of residues is quantitatively less important for binding, but determines specificity by forming base pair specific contacts to three positions in tet operator. This property is most clearly demonstrated for Q38 and P39 and to a lesser extent for T40 at the N-terminus of the recognition alpha-helix. The contacted operator base pairs indicate that the N-terminus of the recognition alpha-helix is located towards the palindromic center in the repressor-operator complex. Although the orientation of the recognition alpha-helix in the Tet repressor-tet operator complex is inversed as compared with the lambda- and 434 repressor-operator complexes, the reduced operator binding of the TA27 mutation in the first alpha-helix suggests that the hydrogen bonding networks connecting the two alpha-helices may be similar in these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

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     

Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding.

We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline.     

Molecular mechanics analysis of Tet repressor TRP-43 fluorescence.

A 35% decrease in the fluorescence intensity of F75 TetR Trp-43 was observed upon binding of the tetracycline derivative 5a,6-anhydrotetracycline (AnTc) to the repressor. The fluorescence decay of Trp-43 in F75 TetR and in its complex with AnTc could be described by the sum of three exponential components, with lifetimes of about 6, 3, and 0.3 ns. The amplitudes, however, were markedly altered upon binding. The minimized energy mapping of Trp-43 chi 1 x chi 2 isomerization clearly indicated the existence of three main potential wells at positions (-160 degrees, -90 degrees) (rotamer I), (-170 degrees, 90 degrees) (rotamer II), and (-70, 150 degrees) (rotamer III). Our study of Trp-43 environment for each of the three rotamers suggests that the longest decay component may be assigned to rotamer II, the middle-lived component to rotamer I, and the subnanosecond component to rotamer III. The origin of the changes in the rotamer distribution upon AnTc binding is discussed. Anisotropy decays are also discussed within the framework of the rotamer model.     

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.     

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.     

Dimerization of the operator binding domain of phage lambda repressor

Dimerization of lambda repressor is required for its binding to operator DNA. As part of a continuing study of the structural basis of the coupling between dimer formation and operator binding, we have undertaken 1H NMR and gel filtration studies of the dimerization of the N-terminal domain of lambda repressor. Five protein fragments have been studied: three are wild-type fragments of different length (1-102, 1-92, and 1-90), and two are fragments bearing single amino acid substitutions in residues involved in the dimer interface (1-102, Tyr-88----Cys; 1-92, Ile-84----Ser). The tertiary structure of each species is essentially the same, as monitored by the 1H NMR resonances of internal aromatic groups. However, significant differences are observed in their dimerization properties. 1H NMR resonances of aromatic residues that are involved in the dimer contact allow the monomer-dimer equilibrium to be monitored in solution. The structure of the wild-type dimer contact appears to be similar to that deduced from X-ray crystallography and involves the hydrophobic packing of symmetry-related helices (helix 5) from each monomer. Removal of two contact residues, Val-91 and Ser-92, by limited proteolysis disrupts this interaction and also prevents crystallization. The Ile-84----Ser substitution also disrupts this interaction, which accounts for the severely reduced operator affinity of this mutant protein.     

Construction of an E. coli strain overproducing the Tn10-encoded TET repressor and its use for large scale purification.

Overproduction of the repressor protein from the Tn10-encoded tetracycline resistance operon is achieved by placement of the respective gene under control of bacteriophage lambda promoter PL in a vector-host system. All cloning steps have to be carried out under repressed conditions to assure survival of the cell. The cI 857 mutation is used to control expression of the tetR gene in large scale fermentation. After induction, the overproducing Escherichia coli strain continues to grow for 2.5 generations before growth terminates. In the expression phase, active TET repressor comprises up to 13% of the total soluble protein. A procedure is described to purify the TET repressor protein to homogeneity on a large scale. Starting from a 10 litre culture, approximately 250 mg of homogeneous, active TET repressor are obtained. The amino acid sequence of the N and C termini are in agreement with the gene start and stop determined from the nucleotide sequence of the Tn10 tetR gene.