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.     

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.     

Interaction of the Escherichia coli Gal repressor protein with its DNA operators in vitro

The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (OE and OI) within the gal operon was analyzed in vitro with quantitative footprint and mobility-shift techniques. In vivo analysis of the regulation of the gal operon [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683-9687] has suggested the role of a regulatory "looped complex" mediated by the association of Gal repressor dimers bound at OE and OI. The binding of Gal repressor to a single site can be described by a model in which monomer and dimer are in equilibrium and only the dimer binds to DNA. At pH 7.0, 25 mM KCl, and 20 degrees C, the binding and dimerization free energies are comparable, suggesting that the equilibrium governing the formation of dimers may be important to regulation. The two intrinsic binding constants, delta GI and delta GE, and a constant describing cooperativity, delta GIE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 degrees C was delta GIE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. delta GE and delta GI had maximal values between pH 6 and pH 7. The dependence of these constants on [KCl] corresponded to the displacement of approximately 2 ion equiv. The temperature dependence could be described by a change in the heat capacity, delta Cp, of -2.3 kcal mol-1 deg-1. Mobility-shift titration experiments conducted at 20 and 0 degrees C yielded values for delta GIE that were consistent with those resolved from the footprint analysis. Unique values of delta GIE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that delta GE = delta GI: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of OE- and OI- operators.     

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.     

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.     

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.     

Lambda cro repressor complex with OR3 operator DNA. 19F nuclear magnetic resonance observations

The interaction of lambda cro repressor with DNA is probed using synthetic 17 base-pair OR3 operators in which 5-fluorodeoxyuridine has been systematically incorporated at each of the nine positions normally occupied by a thymidine residue. By monitoring changes in chemical shift of the fluorine resonances upon cro repressor binding in aqueous buffers of varying 2H2O content, we have examined the specific cro repressor-OR3 DNA complex in detail. The results are interpreted in the context of the popular model for cro repressor-OR3 complex derived from the three-dimensional structure of the cro repressor in the absence of DNA. The results presented here not originally predicted by the model are: (1) there is an asymmetry in the environment at the two ends of the operator, although the base-pairs involved and the cro repressor dimer are symmetric; (2) there appears to be distortion of the DNA helix at two distinct positions; (3) changes of the DNA environment in the middle of the helix suggest additional DNA distortion not near the contact areas proposed in the model.     

Water release associated with specific binding of gal repressor.

Water release coupled to the association of gal repressor with DNA is measured from the sensitivity of the binding constant to the solution osmotic pressure, using neutral solutes that are typically excluded from polar protein and DNA surfaces. Differences in water release for binding of repressor to different sequences are linked with differences in specificity and binding energies. With sucrose, the specific binding of repressor to operator sequences is accompanied by the release of 130 water molecules. No water release is seen for the weak, non-specific binding of repressor to poly(dI-dC).(dI-dC). A difference in the release of six water molecules is seen even for the binding of gal repressor to two different operator sequences that differ in affinity by only a factor of two.     

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.     

Recognition sequences of repressor and polymerase in the operators of bacteriophage lambda

Nucleotide sequences in two wild-type and six mutant operators in the DNA of phage λ are compared. Strikingly similar 17 base pair units are found which we identify as the repressor binding sites. Each operator contains multiple repressor binding sites separated by A-T rich spacers. Elements of 2 fold rotational symmetry are present in each of the sites. Superimposed on each operator is an E. coli RNA polymerase recognition site (promoter). Similarities in the sequences of the two λ promoters, a lac promoter, and an E. coli RNA polymerase recognition site in SV40 DNA are noted.     

Operator DNA sequence variation enhances high affinity binding by hinge helix mutants of lactose repressor protein

The mechanism by which genetic regulatory proteins discern specific target DNA sequences remains a major area of inquiry. To explore in more detail the interplay between DNA and protein sequence, we have examined binding of variant lac operator DNA sequences to a series of mutant lactose repressor proteins (LacI). These proteins were altered in the C-terminus of the hinge region that links the N-terminal DNA binding and core sugar binding domains. Variant operators differed from the wild-type operator, O(1), in spacing and/or symmetry of the half-sites that contact the LacI N-terminal DNA binding domain. Binding of wild-type and mutant proteins was affected differentially by variations in operator sequence and symmetry. While the mutant series exhibits a 10(4)-fold range in binding affinity for O(1) operator, only a approximately 20-fold difference in affinity is observed for a completely symmetric operator, O(sym), used widely in studies of the LacI protein. Further, DNA sequence influenced allosteric response for these proteins. Binding of this LacI mutant series to other variant operator DNA sequences indicated the importance of symmetry-related bases, spacing, and the central base pair sequence in high affinity complex formation. Conformational flexibility in the DNA and other aspects of the structure influenced by the sequence may establish the binding environment for protein and determine both affinity and potential for allostery.     

Two helix DNA binding motif of CAP found in lac repressor and gal repressor.

Comparison of both the DNA and protein sequences of catabolite gene activator protein (CAP) with the sequences of lac and gal repressors shows significant homologies between a sequence that forms a two alpha-helix motif in CAP and sequences near the amino terminus of both repressors. This two-helix motif is thought to be involved in specific DNA sequence recognition by CAP. The region in lac repressor to which CAP is homologous contains many i-d mutations that are defective in DNA binding. Less significant sequence homologies between CAP and phage repressors and activators are also shown. The amino acid residues that are critical to the formation of the two-helix motif are conserved, while those residues expected to interact with DNA are variable. These observations suggest the lac and gal repressors also have a two alpha-helix structural motif which is involved in DNA binding and that this two helix motif may be generally found in many bacterial and phage repressors. We conclude that one major mechanism by which proteins can recognize specific base sequences in double stranded DNA is via the amino acid side chains of alpha-helices fitting into the major groove of B-DNA.     

lac repressor forms loops with linear DNA carrying two suitably spaced lac operators.

Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non-denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a 'tandem' structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a 'sandwich' structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.     

Deoxyribonucleic acid-binding studies on the hut repressor and mutant forms of the hut repressor of Salmonella typhimurium.

In Salmonella typhimurium the genes coding for the enzymes of histidine utilization (hut) are clustered in two adjacent operons, hutMIGC and hut(P,R,Q)UH. A single repressor, the product of the C gene, regulates both operons by binding at two operator sites, one near M and one in (P,R,Q). The deoxyribonucleic acid (DNA)-binding activity of the repressor was measured using DNA's containing separate operators. The repressor had greater activity when assayed using DNA containing the operator of the (P,R,Q)UH operon than when assayed using DNA containing the operator of the MIGC operon. The binding to either operator was absent in the presence of the inducer, urocanate. The DNA-binding activities were also determined for two super-repressors. The super-repressors had altered DNA-binding properties, although the self-regulated nature of the repressors complicated the analysis of the results. A purfication procedure for the wild-type repressor is presented. The purified repressor was somewhat unstable, and additional experiments using it were not performed.