Sequence-specific interactions of the tight-binding I12-X86 lac repressor with non-operator DNA.

The tight-binding I12-X86 lac repressor binds to non-operator DNA in a sequence-specific fashion. Using the DNA of the E. coli I gene we have investigated these sequence-specific interactions and compared them to the operator binding of wild-type repressor. The specific, non-operator DNA interactions are sensitive to the inducer IPTG. One strong binding site in the I gene DNA was found to be one of two expected on the basis of their homology with the lac operator. The binding of I12-X86 repressor to this site was visualized using the footprinting technique, and found to be consistent with an operator-like binding configuration. The protection pattern extends into an adjacent sequence suggesting that two repressor tetramers are bound in tandem.     

lac repressor-operator interaction. Analysis of the X86 repressor mutant

In vitro measurements show that the X86 repressor, which has an increased affinity for the lac operator as compared to wild-type repressor, also has an increased affinity for non-operator sites on Escherichia coli DNA. The rate constant of association of repressor and operator is decreased by E. coli DNA fivefold more for X86 repressor than for wild-type repressor. Low inducer concentrations increase the rate of association of X86 repressor and operator in the presence of E. coli DNA. In a partial equilibrium situation where part of the X86 repressor is bound to the operator, and part to either non-operator sites on E. coli DNA or to an O^c operator, the formation of complexes between X86 repressor and wild-type operator is favored by low inducer concentrations. Repression of the lac enzymes increases drastically in the X86 mutant in the absence of DNA synthesis in vivo. A new explanation for the in vivo characteristics of the X86 mutant is suggested.     

DNA-binding site of lac repressor probed by dimethylsulfate methylation of lac operator.

In order to compare the structures of the DNA-binding sites on variants of the lac repressor, we have studied the influence of these variants on the dimethylsulfate methylation of the lac operator. Since a bound protein changes the availability of specific purines in the operator to this chemical attack, comparisons of the methylation patterns will show similarities or differences in the protein DNA contacts. We compared lac repressor, induced lac repressor (repressor bound to the gratuitous inducer isopropyl-β-d-thiogalactoside), mutant repressors having increased operator affinities (X86, I12 and the X86-I12 double mutant) and repressor peptides (long headpiece, residues 1 to 59 and short headpiece. residues 1 to 51). All of these repressors and repressor peptides exhibit the same general pattern of protection and enhancement in the operator; however, the short headpiece pattern differs most from that of the repressor while the induced repressor and the long headpiece show intermediate patterns that are strikingly similar to each other. The mutant repressors do not show an isopropyl-β-d-thiogalactoside effect but otherwise are almost indistinguishable from wild-type repressor. These results demonstrate that all molecules bind to the operator using basically the same protein-DNA contacts; they imply that (1) most and possibly all repressor contacts to operator lie within amino acids 1 to 51, (2) inducer weakens many contacts rather than totally disrupting one or even a few and (3) the tight-binding mutants do not make additional contacts to the DNA.These results are consistent with a model in which the amino-terminal portions of two repressor monomers make the DNA contacts. We show that one can understand the affinity of binding as related to the accuracy of the register of the two amino-terminal portions along the DNA. Furthermore, the action of inducer and the behaviour of the tight binding mutants can be accomodated within a two-state model in which the strongly or weakly binding states correspond to structures in which the amino-terminal regions are rigidly or loosely held with respect to each other.     

Isolation and characterization of LexA mutant repressors with enhanced DNA binding affinity.

The LexA repressor from Escherichia coli is a sequence-specific DNA binding protein that shows no pronounced sequence homology with any of the known structural motifs involved in DNA binding. Since little is known about how this protein interacts with DNA, we have selected and characterized a great number of intragenic, second-site mutations which restored at least partially the activity of LexA mutant repressors deficient in DNA binding. In 47 cases, the suppressor effect of these mutations was due to an Ind- phenotype leading presumably to a stabilization of the mutant protein. With one exception, these second-site mutations are all found in a small cluster (amino acid residues 80 to 85) including the LexA cleavage site between amino acid residues 84 and 85 and include both already known Ind- mutations as well as new variants like GN80, GS80, VL82 and AV84. The remaining 26 independently isolated second-site suppressor mutations all mapped within the amino-terminal DNA binding domain of LexA, at positions 22 (situated in the turn between helix 1 and helix 2) and positions 57, 59, 62, 71 and 73. These latter amino acid residues are all found beyond helix 3, in a region where we have previously identified a cluster of LexA (Def) mutant repressors. In several cases the parental LexA (Def) mutation has been removed by subcloning or site-directed mutagenesis. With one exception, these LexA variants show tighter in vivo repression than the LexA wild-type repressor. The most strongly improved variant (LexA EK71, i.e. Glu71----Lys) that shows an about threefold increased repression rate in vivo, was purified and its binding to a short consensus operator DNA fragment studied using a modified nitrocellulose filter binding assay. As expected from the in vivo data, LexA EK71 interacts more tightly with both operator and (more dramatically) with non-operator DNA. A determination of the equilibrium association constants of LexA EK71 and LexA wild-type as a function of monovalent salt concentration suggests that LexA EK71 might form an additional ionic interaction with operator DNA as compared to the LexA wild-type repressor. A comparison of the binding of LexA to a non-operator DNA fragment further shows that LexA interacts with the consensus operator very selectively with a specificity factor of Ks/Kns of 1.4 x 10(6) under near-physiological salt conditions.     

Hinge-helix formation and DNA bending in various lac repressor-operator complexes

The hinge-region of the lac repressor plays an important role in the models for induction and DNA looping in the lac operon. When lac repressor is bound to a tight-binding symmetric operator, this region forms an alpha-helix that induces bending of the operator. The presence of the hinge-helices is questioned by previous data that suggest that the repressor does not bend the wild-type operator. We show that in the wild-type complex the hinge-helices are formed and the DNA is bent, similar to the symmetric complex. Furthermore, our data show differences in the binding of the DNA binding domains to the half-sites of the wild-type operator and reveal the role of the central base-pair of the wild-type operator in the repressor-operator interaction. The differences in binding to the operator half-sites are incorporated into a model that explains the relative affinities of the repressor for various lac operator sequences that contain left and right half-sites with different spacer lengths.     

Mutant λ Repressors with Increased Operator Affinities Reveal New, Specific Protein-DNA Contacts

The binding specificities of four mutant lambda cI repressor proteins with increased affinities for operator DNA were examined. Two mutant repressors (Glu34----Lys and Glu83----Lys) have the same specificity of binding as wild-type repressor, whereas two (Gly48----Ser and Gly48----Asn) have new binding specificities. The Gly48----Asn mutant repressor recognizes lambda operators with changes at base pair 3 with a different order of affinity than wild-type repressor, suggesting that the side chain of Asn48 makes additional specific DNA contacts at or near this base pair. When paired with a change that disrupts the specific interaction of the amino-terminal arm of lambda repressor with DNA (Lys4----Gln), one change that increases the affinity of repressor (Gly48----Ser) suppresses the binding defect of the Lys4----Gln repressor, resulting in a double mutant repressor with a new binding specificity different than that of both its parents and of wild type. These results lend strong support to the model of direct recognition of the lambda operator by lambda repressor proposed from the crystal structure of the repressor/operator complex.     

Modular construction of extended DNA recognition surfaces: mutant DNA-binding domains of the 434 repressor as building blocks

Single-chain derivatives of the 434 repressor containing one wild-type and one mutant DNA-binding domain recognize the general operator ACAA-6 base pairs-NNNN, where the ACAA operator subsite is contacted by the wild-type and the NNNN tetramer by the mutant domain. The DNA-binding specificities of several single-chain mutants were studied in detail and the optimal subsites of the mutant domains were determined. The characterized mutant domains were used as building units to obtain homo- and heterodimeric single-chain derivatives. The DNA-binding properties of these domain-shuffled derivatives were tested with a series of designed operators of NNNN-6 base pairs-NNNN type. It was found that the binding specificities of the mutant domains were generally maintained in the new environments and the binding affinities for the optimal DNA ligands were high (with K(d) values in the range of 10(-11)-10(-10) M). Considering that only certain sequence motifs in place of the six base pair spacer can support optimal contacts between the mutant domains and their subsites, the single-chain 434 repressor mutants are highly specific for a limited subset of 14 base pair long DNA targets.     

Repression of the E coli recA gene requires at least two LexA protein monomers

To analyze the DNA binding domain of E coli LexA repressor and to test whether the repressor binds as a dimer to DNA, negative dominant lexA mutations affecting the binding domain have been isolated. A large number of amino acid substitutions between amino acid positions 39 and 46 were introduced using cassette mutagenesis. Mutants defective in DNA binding were identified and then examined for dominance to lexA+. A number of substitutions weakened repressor function partially, whereas other substitutions led to a repressor with no demonstrable activity and a defective dominant phenotype. Since the LexA binding site has dyad symmetry, we infer that this dominance results from interaction of monomers of wild-type LexA protein with mutant monomers and that an oligomeric form of repressor binds to operator. The binding of LexA protein to operator DNA was investigated further using a mutant protein, LexA408, which recognizes a symmetrically altered operator mutant but not wild-type operator. A mixture of mutant LexA408 and LexA+ proteins, but neither individual protein, bound to a hybrid recA operator consisting of mutant and wild-type operator half sites. These results suggest that at least 1 LexA protein monomer interacts with each operator half site. We discuss the role of LexA oligomer formation in binding of LexA to operator DNA.     

The basis for the super-repressor phenotypes of the AV77 and EK18 mutants of trp repressor

The DNA-binding properties of two super-repressor mutants of the Escherichia coli trp repressor, EK18 and AV77, have been investigated using steady-state fluorescence anisotropy measurements, in order to further elucidate the basis for their super-repressor phenotypes. Several suggestions have been previously proposed as the basis for the super-repressor phenotype of EK18 and AV77. For the negative to positive charge change EK18 mutant, increased electrostatic interactions between the EK18 mutant and the operator and increased protein-protein interactions between EK18 dimers have been suggested as contributing to the super-repressor phenotype of this mutant. We show that EK18 dimers actually bind to wild-type and variant operator sequences with a decrease in apparent cooperativity and an increase in affinity, compared to WTTR dimers. Thus, the EK18 super-repressor phenotype is not due to increased cooperative binding between EK18 dimers. These results support the hypothesis that the super-repressor phenotype of EK18 arises from increased electrostatic interactions between the mutant and DNA. In the case of the AV77 mutant, weaker binding affinity of apo-AV77 to non-specific DNA, increased selectivity of binding of AV77 for the operator, and a higher population of folded functional AV77 dimers available to bind the operator under limiting L-Trp conditions in vivo, have been proposed for the super-repressor phenotype of this mutant. We show that like the EK18 mutant, apoAV77 binds with higher affinity to non-specific DNA compared to apo-WTTR and that the holo-AV77 mutant does not bind with higher selectivity to the operator, has had been previously proposed. We therefore conclude that the super-repressor phenotype of the AV77 mutant is due to an increase in the population of folded, functional AV77 dimers, under limiting L-Trp conditions in vivo.     

Evidence for leucine zipper motif in lactose repressor protein

Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.     

Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants.

We have analyzed the DNA binding properties of Tet-repressor mutants with single amino acid residue replacements at eight positions within the alpha-helix-turn-alpha-helix DNA-binding motif. A saturation mutagenesis of Gln38, Pro39, Thr40, Tyr42, Trp43 and His44 in the second alpha-helix was performed; in addition, several substitutions of Thr27 and Arg28 in the first alpha-helix were constructed. The abilities of these mutant repressors to bind a set of 16 operator variants were determined and revealed 23 new binding specificities. All repressor mutants with DNA-binding activity were inducible by tetracycline, while mutants lacking binding activity were trans-dominant over the wild-type. All mutant proteins were present at the same intracellular steady-state concentrations as the wild-type. These results suggest the structural integrity of the mutant repressors. On the basis of the new recognition specificities, five contacts between a repressor monomer and each operator half-site and the chemical nature of these repressor-operator interactions are proposed. We suggest that Arg28 contacts guanine of the G.C base-pair at operator position 2 with two H-bonds, Gln38 binds adenine of the A.T base-pair at position 3 with two H-bonds, and the methyl group of Thr40 participates in a van der Waals' contact with cytosine of the G.C base-pair at position 6 of tet operator. A previously unrecognized type of interaction is proposed for Pro39, which inserts its side-chain between the methyl groups of the thymines of T.A and A.T base-pairs at positions 4 and 5. Computer modeling of these proposed contacts reveals that they are possible using the canonical structures of the helix-turn-helix motif and B-DNA. These contacts suggest an inverse orientation of the Tet repressor helix-turn-helix with respect to the operator center as compared with non-inducible repressor-operator complexes, and are supported by similar contacts of other repressor-operator complexes.     

Effect of amino acid alterations in the tryptophan-binding site of the trp repressor

Mutations in the tryptophan-binding site of the trp repressor have been generated using site-directed mutagenesis. The selection of sites for alteration was based on the three-dimensional x-ray crystallographic structure (Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L., and Sigler, P. B. (1985) Nature 317, 782-786). The changes generated include Thr-44 to Ala (T44A), Arg-54 to Leu (R54L), Arg-54 to Lys (R54K), Arg-84 to Leu (R84L), and Arg-84 to Lys (R84K). The mutant proteins were purified and characterized in detail for their binding properties. Both tryptophan and operator DNA affinities for all five mutants were decreased. The R84L, R54K, and R54L mutants exhibited increases in Kd for operator DNA relative to wild-type repressor ranging from approximately 10(3) to approximately 10(4), while R84K and T44A exhibited increases of 10- to 100-fold. This diminution in DNA binding activity derives at least in part from diminished affinity for tryptophan, although decreased affinity for nonspecific DNA was also observed for these mutant proteins. Tryptophan binding was not detectable by equilibrium dialysis for most of the mutant proteins, but this activity was measurable for several of the altered proteins by monitoring the fluorescence decrease associated with the displacement of 1-anilino-8-naphthalenesulfonate from the tryptophan-binding site (Chou, W.-Y., and Matthews, K. S. (1989) J. Biol. Chem. 264, 18314-18319). These measurements revealed that tryptophan bound to R84K, T44A, and R84L repressors with Kd values 1.5- to 13-fold higher than that for wild-type repressor. It was not possible to detect tryptophan binding to R54K and R54L even using the fluorescence assay. Circular dichroism spectra demonstrated that the mutants and the wild-type repressor possess similar secondary structural features. The results of this selected substitution in the tryptophan-binding site are readily interpreted based on the x-ray structural analysis.     

Wrapping of flanking non-operator DNA in lac repressor-operator complexes: implications for DNA looping

In our studies of lac repressor tetramer (T)-lac operator (O) interactions, we observed that the presence of extended regions of non-operator DNA flanking a single lac operator sequence embedded in plasmid DNA produced large and unusual cooperative and anticooperative effects on binding constants (Kobs) and their salt concentration dependences for the formation of 1:1 (TO) and especially 1:2 (TO2) complexes. To explore the origin of this striking behavior we report and analyze binding data on 1:1 (TO) and 1:2 (TO2) complexes between repressor and a single O(sym) operator embedded in 40 bp, 101 bp, and 2514 bp DNA, over very wide ranges of [salt]. We find large interrelated effects of flanking DNA length and [salt] on binding constants (K(TO)obs, K(TO2)obs) and on their [salt]-derivatives, and quantify these effects in terms of the free energy contributions of two wrapping modes, designated local and global. Both local and global wrapping of flanking DNA occur to an increasing extent as [salt] decreases. Global wrapping of plasmid-length DNA is extraordinarily dependent on [salt]. We propose that global wrapping is driven at low salt concentration by the polyelectrolyte effect, and involves a very large number (>/similar 20) of coulombic interactions between DNA phosphates and positively charged groups on lac repressor. Coulombic interactions in the global wrap must involve both the core and the second DNA-binding domain of lac repressor, and result in a complex which is looped by DNA wrapping. The non-coulombic contribution to the free energy of global wrapping is highly unfavorable ( approximately +30-50 kcal mol(-1)), which presumably results from a significant extent of DNA distortion and/or entropic constraints. We propose a structural model for global wrapping, and consider its implications for looping of intervening non-operator DNA in forming a complex between a tetrameric repressor (LacI) and one multi-operator DNA molecule in vivo and in vitro. The existence of DNA wrapping in LacI-DNA interactions motivates the proposal that most if not all DNA binding proteins may have evolved the capability to wrap and thereby organize flanking regions of DNA.     

Mutational analysis of the NH2-terminal arms of the trp repressor indicates a multifunctional domain

The NH₂-terminal arms of the Escherichia coli trp repressor have been implicated in three functions: formation of repressor–operator complexes via association with non-operator DNA; stabilization of repressor oligomers bound to DNA; and oligomerization of the aporepressor in the absence of DNA. To begin to examine the structural aspects of the arms that are responsible for these varied activities, we generated an extensive set of deletion and substitution mutants and measured the activities of these mutants in vivo using reporter gene fusions. Deletion of any part of the arms resulted in a significant decrease in repressor activity at both the trp and the trpR operons. Positions 4, 5 and 6 were the most sensitive to missense changes. Most substitutions at these positions resulted in repressors with less than 5% of the activity of the wild-type trp repressor. A large percentage of the missense mutants were more active than the wild-type repressor in medium containing tryptophan and less active in medium without tryptophan. This phenotype can be explained in terms of altered oligomerization of both the repressor and the aporepressor. Also, nine super-repressor mutants, resulting from substitutions clustered at both ends of the arms, were found. Our results support the hypothesis that the NH₂-terminal arm of the trp repressor is a multifunctional domain and reveal structural components likely to be involved in the various functions.     

Phage λ Cro Protein and Ci Repressor Use Two Different Patterns of Specific Protein-DNA Interactions to Achieve Sequence Specificity in Vivo

By assaying the binding of wild-type Cro to a set of 40 mutant lambda operators in vivo, we have determined that the 14 outermost base pairs of the 17 base pair, consensus lambda operator are critical for Cro binding. Cro protein recognizes 4 base pairs in a lambda operator half-site in different ways than cI repressor. The sequence determinants of Cro binding at these critical positions in vivo are nearly perfectly consistent with the model proposed by W. F. ANDERSON, D. H. OHLENDORF, Y. TAKEDA and B. W. MATTHEWS and modified by Y. TAKEDA, A. SARAI and V. M. RIVERA for the specific interactions between Cro and its operator, and explain the relative order of affinities of the six natural lambda operators for Cro. Our data call into question the idea that lambda repressor and Cro protein recognize the consensus lambda operator by nearly identical patterns of specific interactions.     

Affinities of tight-binding lactose repressors for wild-type and pseudo-operators

Five tight-binding (Itb) mutants of the Escherichia coli lactose (lac) repressor have been characterized with regard to their non-specific affinity for DNA and their specific affinity for the wild-type operator and several sequence-altered (pseudo-) operators. Repressor-operator association rates were determined in the presence or absence of competitor DNA, dissociation rates of repressor from various DNA fragments were measured, and equilibrium competition for repressor binding was examined for several pseudo-operator DNAs. The mutant repressors exhibited increased non-specific affinity for DNA, and variable increases in affinity for sequence-altered operators. The known positions of amino acid substitutions for three of these Itb repressors support suggestions that residues 51 to 64 are important for operator recognition in addition to residues 1 to 50.     

DNA Sequence Determinants of λ Repressor Binding in Vivo

The critical operator determinants for λ repressor recognition have been defined by analyzing the binding of wild-type repressor to a set of mutant operators in vivo. Base pair substitutions at six positions within the λ operator half-site impair binding severely, and define these base pairs as critical for operator function. One mutant operator binds repressor better than the consensus operator, and is a superoperator. The model proposed by M. Lewis in 1983 for the binding of λ repressor to its operator accurately predicts the observed operator requirements for binding in vivo, with several minor exceptions. The order of affinities of the six natural λ operators has also been determined.     

Role of the Cro repressor carboxy-terminal domain and flexible dimer linkage in operator and nonspecific DNA binding

A series of mutations comprising single and multiple substitutions, deletions, and extensions within the carboxy-terminal domain of the bacteriophage lambda Cro repressor have been constructed. These mutations generally affect the affinity of repressor for specific and nonspecific DNA. Additionally, substitution of the carboxy-terminal alanine with several amino acids capable of hydrogen-bonding interactions leads to improved specific binding affinities. A mutation is also described whereby cysteine links the two Cro monomers by a disulfide bond. As a consequence, a significant improvement in nonspecific binding and a concomitant reduction in specific binding are observed with this mutant. These results provide evidence that the carboxy terminus of Cro repressor is an important DNA binding domain and that a flexible connection between the two repressor monomers is a critical factor in modulating the affinity of wild-type repressor for DNA.     

P22 repressor mutants deficient in co-operative binding and DNA loop formation.

We show here, both in vivo and in vitro, that P22 repressor binds co-operatively to operator sites separated by an integral number of turns of the DNA helix. We measure this co-operativity in vivo using an assay in which repression of a promoter requires co-operative binding of P22 repressors to two separated (non-adjacent) operator sites. We report the isolation of mutant repressors that have high affinity for single operator sites, but are defective in co-operative binding. Six different mutants, all bearing single amino acid changes in the carboxyl domain, have been isolated. We purified the two mutants most deficient in co-operative binding, and found that they bind non-co-operatively in vitro to adjacent as well as to non-adjacent pairs of operator sites.