DNA conformational change in Gal repressor-operator complex: involvement of central G-C base pair(s) of dyad symmetry.

Gal repressor dimer binds to two gal operator sites, OE and OI, which are 16 bp long similar sequences with hyphenated dyad symmetries (11,12). Repressor occupation hinders the reactivity of the N7 atoms in the major groups of guanines, located at positions 1, 3 and 8, and the rotational 1', 3' and 8' of the symmetries. We have shown that Gal repressor binding to OE or OI DNA fragments increases the circular dichroism (CD) spectral peak in the 270 to 300 nm range. The CD change is similar to that observed for Lac repressor binding to its operator site (14). It is consistent with a DNA conformational change during complex formation between Gal repressor and OE and OI DNA. The CD spectral change was not observed when the central 8,8' G-C base pairs in the DNA-protein complex were replaced by A-T base pairs, whereas substitution of the 1,1' G-C base pairs do show the accompanying increase in the spectra during repressor binding. The absence of CD change of the Gal repressor complex with DNA mutated at the 8,8' base pairs suggest that the central G-C base pairs are required for the repressor induced conformational change.     

Stability of a Lac repressor mediated "looped complex"

The quantitation of the stability of a protein-mediated "looped complex" of the Lac repressor and DNA containing two protein-binding sites whose centers of symmetry are separated by 11 helical turns (114 bp) was accomplished by footprint and gel mobility-shift titration techniques. Lac repressor binding to this DNA was only moderately cooperative; a cooperative free energy of -1.0 kcal/mol was calculated in a model-independent fashion from the individual-site loading energies obtained from the footprint titration studies. In order to partition the cooperative binding energy into components representing the dimer-tetramer association of Lac repressor and the cyclization probability of the intervening DNA, advantage was taken of the presence of experimental measures that were in proportion to the concentration of the looped complex present in solution. One measure was the DNase I hypersensitivity observed in footprint titrations in bands located between the two binding sites. The second measure resulted from the electrophoretic resolution in the gel mobility-shift titrations of the band representing the doubly liganded "tandem complex" from the band representing the singly liganded complexes, including the looped complex. Analysis of the footprint and mobility-shift titration data utilizing this additional information showed that approximately 65% of the molecules present in solution are looped complexes at pH 7.0, 100 mM KCl, and 20 degrees C when the binding sites on the DNA are saturated with protein. Reconciliation of the observed low binding cooperativity and the high proportion of looped complexes could only be obtained when the titration data were analyzed by a model in which Lac repressor tetramers dissociate into dimers in solution. The proportion of looped complexes present in solution is highly dependent on the dimer-tetramer association constant, delta Gtet. This result is consistent with the determination by high-pressure fluorescence techniques that Lac repressor tetramers dissociate with an association free energy comparable to their DNA-binding free energies [Royer, C. A., Chakerian, A. E., & Matthews, K. S. (1990) Biochemistry 29, 4959-4966]. However, when the value of delta Gtet of -10.6 kcal/mol (at 20 degrees C) reported by Royer et al. (1990) is assumed, the titration data demand that tetramers bind DNA with much greater affinity than dimers: a result inconsistent with the destabilization of tetramers by the operator observed in the dimer-tetramer dissociation studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of HU and DNA supercoiling in transcription repression: specialized nucleoprotein repression complex at gal promoters in Escherichia coli

Efficient repression of the two promoters P1 and P2 of the gal operon requires the formation of a DNA loop encompassing the promoters. In vitro, DNA looping-mediated repression involves binding of the Gal repressor (GalR) to two gal operators (OE and OI) and binding of the histone-like protein HU to a specific locus (hbs) about the midpoint between OE and OI, and supercoiled DNA. Without DNA looping, GalR binding to OE partially represses P1 and stimulates P2. We investigated the requirement for DNA supercoiling and HU in repression of the gal promoters in vivo in strains containing a fusion of a reporter gene, gusA or lacZ, to each promoter individually. While the P1 promoter was found to be repressible in the absence of DNA supercoiling and HU, the repression of P2 was entirely dependent upon DNA supercoiling in vivo. The P2 promoter was fully derepressed when supercoiling was inhibited by the addition of coumermycin in cells. P2, but not P1, was also totally derepressed by the absence of HU or the OI operator. From these results, we propose that the repression of the gal promoters in vivo is mediated by the formation of a higher order DNA-multiprotein complex containing GalR, HU and supercoiled DNA. In the absence of this complex, P1 but not P2 is still repressed by GalR binding to OE. The specific nucleoprotein complexes involving histone-like proteins, which repress promoter activity while remaining sensitive to inducing signals, as discussed, may occur more generally in bacterial nucleoids.     

Probing the structure of gal operator-repressor complexes. Conformation change in DNA

The gal operon is regulated by binding of Gal repressor to two operator loci, OE and OI, which are separated by 114 base pairs (bp). We have probed the actual operator DNA segments with and without Gal repressor occupation by characterizing the regions protected by repressor from DNase I digestion and dimethyl sulfate methylation. The segments which are protected from DNase I digestion in both OE and OI are about 22 bp long and seem to include 2-3 extra bp on either side of a 16-bp similar sequence containing an approximate dyad symmetry, with a consensus half-symmetry sequence GTG(G/T)AA-C. Repressor occupation hinders the reactivity of the consensus guanines in the four half-symmetry sequences, as shown by retardation of methylation at the N-7 positions by dimethyl sulfate owing to repressor binding. The protected guanines are symmetrically located. Since a dimeric Gal repressor affects symmetrically located bases, it is consistent with the notion that each half-operator is occupied by a repressor subunit. Because the N-7 positions of methylation of guanines lie in the major grooves and the protected guanines are located at positions 1, 3, 8 and the rotational 1', 3', and 8' in the 16-bp dyad symmetry, we suggest that Gal repressor establishes direct contacts with bases at 1, 3, 1', and 3' through two major grooves lying on one face of an operator helix and prevents reactivity of the guanines at 8 and 8' of a third major groove on the opposite face by changing the DNA helical structure at this position. Contacts at other positions are also discussed.     

Purification and properties of Gal repressor:pL-galR fusion in pKC31 plasmid vector

The galR gene, which encodes the Gal repressor protein in Escherichia coli, has been fused to the strong pL promoter of bacteriophage lambda in plasmid pKC31. The pL promoter is kept repressed by a thermolabilie lambda repressor, CIts857, to prevent cell killing. Heat induction of the pL-galR fusion plasmid synthesizes large amounts of active Gal repressor. The protein has been purified to homogeneity in three steps. The purification is greatly aided by the reversible insolubility of active repressor in crude extract at salt concentrations of less than 200 mM. The amino-terminal amino acid sequence determined by automated Edman degradation is: N-Ala-Thr-Ile-Lys-Asp-Val-Ala-Arg-Leu-Ala-Gly-Val-Ser-Val-Ala-Thr-Val-. Comparison of this sequence with that deduced from the DNA sequence of the galR gene showed that the formyl methionine residue preceding alanine at position 1 is cleaved off. The repressor is present in solution as a dimer of a 37-kDa subunit. The protein binds to gal DNA containing wild type and not mutant operator sequences. As predicted, this sequence-specific binding is inhibited by the presence of D-galactose or D-fucose, both of which are in vivo inducers of the gal operon. Gal repressor inhibits the expresison of gal operon by binding to two spatially separated operators which flank, but do not overlap, the gal promoter segment. Experiments to study the mechanism of repressor action are discussed.     

A control element within a structural gene: the gal operon of Escherichia coli

The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2. Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus. Both the promoters are negatively regulated by a single repressor, the product of the galR gene. An operator site, defined by several mutations, has previously been located upstream from the cat locus. We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing. From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene. Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2. This would be the first example of the presence of a functional operator element within a structural protein-coding region.     

Cooperative protein-DNA interactions: effects of KCl on lambda cI binding to OR.

The effects of monovalent salt activity on the site-specific and cooperative interactions of cI repressor with its three operator sites OR were studied by using quantitative DNase I footprint titration methods. Individual-site binding isotherms were obtained for binding repressor dimers to each site of wild-type OR and to mutant operator templates in which binding to one or two sites has been eliminated. The standard Gibbs energies for intrinsic binding, delta G1, delta G2, and delta G3, and cooperative interactions, delta G12 and delta G23, were determined at each condition (range 50-200 mM KCl). It is found that the dimer affinity for each of the three sites increases as [KCl] decreases, a striking result given that the monomer-dimer equilibrium shifts toward monomer formation under identical solution conditions [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry (preceding paper in this issue)]. The magnitudes of ion-linked effects are found to differ at the three operator sites, while the intrinsic interaction binding free energies for sites OR1 and OR3 change in parallel over the entire range of [KCl]. The KCl dependencies at OR1 and OR3 represent the average release of 3.7 +/- 0.6 and 3.8 +/- 0.6 apparent ions, respectively. By contrast, the KCl dependency of OR2 binding corresponds to the displacement of 5.2 +/- 0.7 apparent ions. The ability of cI repressor to discriminate between the three operator sites thus appears linked to ion binding/release reactions.     

DNA-binding properties of a lac repressor mutant incapable of forming tetramers

The interaction of proteins bound to sites widely separated on the genome is a recurrent motif in both prokaryotic and eukaryotic regulatory systems. Lac repressor mediates the formation of "DNA loops" by the simultaneous interaction of a single protein tetramer with two DNA-binding sites. The DNA-binding properties of a Lac repressor mutant (LacIadi) deficient in the association of protein dimers to tetramers was investigated. The results of quantitative footprint and gel mobility-shift titrations suggest that the wild-type Lac repressor (LacI+) binds cooperatively to two operator sites separated by 11 helical turns on a linear DNA restriction fragment by the formation of a "looped complex." LacIadi binds to this two-site operator non-cooperatively and without formation of a looped complex. These results demonstrate that the dimer-tetramer association of LacI+ is directly responsible for its cooperative binding and its ability to mediate formation of a looped complex. The Iadi mutation disrupts the monomer-dimer as well as eliminating the dimer-tetramer association equilibria while the DNA binding affinity of LacIadi to a single site is unchanged relative to the wild-type protein. These results suggest that DNA binding and dimer-tetramer association are functionally unlinked. The similarity of the DNA-binding properties of LacIadi and Gal repressor, a protein believed to function by mediating the formation of a looped complex, are discussed.     

Ligand specificity and ligand-induced conformational change in gal repressor

Gal repressor (GalR) binds D-galactose, which is responsible for lifting of repression of the gal operon. Proton T1 measurements of alpha- and beta-anomers of galactose as a function of gal repressor show preferential binding of the beta-anomer. The beta-anomer was isolated by high-performance liquid chromatography and was shown to bind tightly to GalR. Calorimetry was used to determine enthalpy changes at several temperatures. Heat capacity change was found to be positive, indicating that a significant amount of hydrophobic surface area was exposed upon galactose binding. Bis-ANS binding to GalR is significantly enhanced in the presence of a saturating amount of galactose, indicating additional exposure of hydrophobic surfaces. We propose that the galactose-induced conformational change involves the opening of the two subdomains, which may disrupt protein-protein interactions responsible for repression.     

Regulation of open complex formation at the Escherichia coli galactose operon promoters. Simultaneous interaction of RNA polymerase, gal repressor and CAP/cAMP.

The regulation of open complex formation at the Escherichia coli galactose operon promoters by galactose repressor and catabolite activator protein/cyclic AMP (CAP/cAMP) was investigated in DNA-binding and kinetic experiments performed in vitro. We found that gal repressor and CAP/cAMP bind to the gal regulatory region independently, resulting in simultaneous occupancy of the two gal operators and the CAP/cAMP binding site. Both CAP/cAMP and gal repressor altered the partitioning of RNA polymerase between the two overlapping gal promoters. Open complexes formed in the absence of added regulatory proteins were partitioned between gal P1 and P2 with occupancies of 25% and 75%, respectively. CAP/cAMP caused open complexes to be formed nearly exclusively at P1 (98% occupancy). gal repressor caused a co-ordinated, but incomplete, switch in promoter partitioning from P1 to P2 in both the absence and presence of CAP/cAMP. We measured the kinetic constants governing open complex formation and decay at the gal promoters in the absence and presence of gal repressor and CAP/cAMP. CAP/cAMP had the largest effect on the kinetics of open complex formation, resulting in a 30-fold increase in the apparent binding constant. We conclude that the regulation of open complex formation at the gal promoters does not result from competition between gal repressor, CAP/cAMP and RNA polymerase for binding at the gal operon regulatory region, but instead results from the interactions of the three proteins during the formation of a nucleoprotein complex on the gal DNA fragment. Finally, we present a kinetic model for the regulation of open complex formation at the gal operon.     

Site-specific enthalpic regulation of DNA transcription at bacteriophage lambda OR.

Binding of cI repressor to DNA fragments containing the three specific binding sites of the right operator (OR) of bacteriophage lambda was studied in vitro over the temperature range 5-37 degrees C by quantitative footprint titration. The individual-site isotherms, obtained for binding repressor dimers to each site of wild-type OR and to appropriate mutant operator templates, were analyzed for the Gibbs energies of intrinsic binding and pairwise cooperative interactions. It is found that dimer affinity for each of the three sites varies inversely with temperature, i.e., the binding reactions are enthalpy driven, unlike many protein-DNA reactions. By contrast, the magnitude of the pairwise cooperativity terms describing interaction between adjacently site-bound repressor dimers is quite small. This result in combination with the recent finding that repressor monomer-dimer assembly is highly enthalpy driven (with delta H degrees = -16 kcal mol-1) [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry 30, 7817-7821] indicates that the associative contacts between site-bound repressors that mediate cooperativity are unlikely to be the same as those responsible for dimerization. The intrinsic binding enthalpies for all three sites are negative (exothermic) and nearly temperature-invariant, indicating no heat capacity changes on the scale of those inferred in other protein-DNA systems. However, the three operator sites are affected differentially by temperature: the intrinsic binding free energies for sites OR1 and OR3 change in parallel over the entire range, delta H0OR1 = -23.3 +/- 4.0 kcal mol-1 and delta H0OR3 = -22.7 +/- 1.2 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA polymerase and gal repressor bind simultaneously and with DNA bending to the control region of the Escherichia coli galactose operon.

The Escherichia coli galactose operon contains an unusual array of closely spaced binding sites for proteins governing the expression from the two physically overlapping gal promoters. Based on studies of two gal promoter-up mutants we have previously suggested RNA-polymerase-induced DNA bending of gal promoter DNA. Here we present new evidence confirming and extending this interpretation. It was obtained by the circular permutation assay of gel electrophoretic mobility [Wu and Crothers (1984), Nature, 308, 509-513] applied to three analogous series of circularly permuted fragments derived from wild-type and two promoter-up mutant DNAs. The same circularly permuted DNA fragments have further been used to study the binding of gal repressor to its operator sites by electrophoretic mobility shift and by DNase I footprinting techniques. The main results are: (i) complexes carrying repressor either exclusively at the upstream operator O1 or at the downstream operator O2 exhibit different electrophoretic mobilities; (ii) binding to either one of the operators results in protein-induced DNA bending by the criteria of the circular permutation mobility assay; and (iii) occupation of both gal operators by gal repressor does not prevent cAMP-CRP-independent binding of RNA polymerase to the gal promoters, as judged by DNase I protection and gel retardation assays. The latter finding imposes constraints on any attempt to model the regulation of gal expression by assumed DNA-protein and protein-protein interactions.     

72 residues of gal repressor fused to beta-galactosidase repress the gal operon of E. coli

An active gene has been constructed which produces a chimera consisting of the N-terminal domain of the gal repressor and all but the first five residues of beta-galactosidase. Seventy two residues of gal repressor fused to beta-galactosidase as tetrameric core are sufficient to repress the gal operon in vivo and to bind to the gal operator in vitro.