Operator sequence alters gene expression independently of transcription factor occupancy in bacteria.

A canonical quantitative view of transcriptional regulation holds that the only role of operator sequence is to set the probability of transcription factor binding, with operator occupancy determining the level of gene expression. In this work, we test this idea by characterizing repression in vivo and the binding of RNA polymerase in vitro in experiments where operators of various sequences were placed either upstream or downstream from the promoter in Escherichia coli. Surprisingly, we find that operators with a weaker binding affinity can yield higher repression levels than stronger operators. Repressor bound to upstream operators modulates promoter escape, and the magnitude of this modulation is not correlated with the repressor-operator binding affinity. This suggests that operator sequences may modulate transcription by altering the nature of the interaction of the bound transcription factor with the transcriptional machinery, implying a new layer of sequence dependence that must be confronted in the quantitative understanding of gene expression.

Crystal structure of TtgV in complex with its DNA operator reveals a general model for cooperative DNA binding of tetrameric gene regulators.

The majority of bacterial gene regulators bind as symmetric dimers to palindromic DNA operators of 12-20 base pairs (bp). Multimeric forms of proteins, including tetramers, are able to recognize longer operator sequences in a cooperative manner, although how this is achieved is not well understood due to the lack of complete structural information. Models, instead of structures, of complete tetrameric assembly on DNA exist in literature. Here we present the crystal structures of the multidrug-binding protein TtgV, a gene repressor that controls efflux pumps, alone and in complex with a 42-bp DNA operator containing two TtgV recognition sites at 2.9 Å and 3.4 Å resolution. These structures represent the first full-length functional tetrameric protein in complex with its intact DNA operator containing two continuous recognition sites. TtgV binds to its DNA operator as a highly asymmetric tetramer and induces considerable distortions in the DNA, resulting in a 60° bend. Upon binding to its operator, TtgV undergoes large conformational changes at the monomeric, dimeric, and tetrameric levels. The structures here reveal a general model for cooperative DNA binding of tetrameric gene regulators and provide a structural basis for a large body of biochemical data and a reinterpretation of previous models for tetrameric gene regulators derived from partial structural data.

Heme-responsive DNA binding by the global iron regulator Irr from Rhizobium leguminosarum.

Heme, a physiologically crucial form of iron, is a cofactor for a very wide range of proteins and enzymes. These include DNA regulatory proteins in which heme is a sensor to which an analyte molecule binds, effecting a change in the DNA binding affinity of the regulator. Given that heme, and more generally iron, must be carefully regulated, it is surprising that there are no examples yet in bacteria in which heme itself is sensed directly by a reversibly binding DNA regulatory protein. Here we show that the Rhizobium leguminosarum global iron regulatory protein Irr, which has many homologues within the alpha-proteobacteria and is a member of the Fur superfamily, binds heme, resulting in a dramatic decrease in affinity between the protein and its cognate, regulatory DNA operator sequence. Spectroscopic studies of wild-type and mutant Irr showed that the principal (but not only) heme-binding site is at a conserved HXH motif, whose substitution led to loss of DNA binding in vitro and of regulatory function in vivo. The R. leguminosarum Irr behaves very differently to the Irr of Bradyrhizobium japonicum, which is rapidly degraded in vivo by an unknown mechanism in conditions of elevated iron or heme, but whose DNA binding affinity in vitro does not respond to heme.

[Statistical fluctuations of the filling level of operator by repressor determine the level of noise of reporter gene expression].

The regulation of the reporter gene activity in a single bacterial cell by means of lambda-phage C1 repressor has been described by the methods of statistical thermodynamics. The equations for the calculation of the mean production rate of the reporter protein and its standard deviation as a function of C1 repressor concentration in the cell have been obtained. The stochastic nature of C1 repressor binding with OR1 and OR2 operator sites becomes apparent when both repressor molecules and operators are present in the bacterial cell in a small number of copies. In this case, the number of repressor molecules that bind to OR1 and OR2 sites fluctuates considerably. The in vitro binding of C1 repressor to OR1 and OR2 sites, their mutant forms, and nonspecific DNA regions has been well studied. Using the binding constants of in vitro binding of C1 repressor to OR1, OR2 and nonspecific DNA regions and also the value of the cooperativity parameter for C1 repressor binding to OR1 and OR2 sites, we calculated the mean rate of synthesis of the reporter protein and its standard deviation as a function of repressor concentration in cell. The theoretical relations fit well the experimental results. The results of calculations confirm the assumption that gene expression noise in a single cell at a repressor concentration exceeding 100 nM is related to the stochastic nature of binding of repressor dimers to OR1 and OR2 sites. Other mechanisms of the generation of gene expression noise (for example, monomer-dimer balance) make a significant contribution at concentrations less than 100 nM.

Feedback regulation of Lac repressor expression in Escherichia coli.

Negative feedback regulation, mediated through repressor binding site O3, which overlaps the lacI gene, could explain the robustness of the weak expression of Lac repressor. Significant autorepression of Lac repressor has never been ruled out. In the work presented here, the degree of autoregulation of Lac repressor was determined. It is negligible.

Optimization of the palindromic order of the TtgR operator enhances binding cooperativity.

TtgR is the specific transcriptional repressor of the TtgABC efflux pump. TtgR and the TtgB efflux pump proteins possess multidrug-binding capacity, and their concerted action is responsible for the multidrug resistance phenotype of Pseudomonas putida DOT-T1E. TtgR binds to a pseudo-palindromic site that overlaps the ttgR/ttgA promoters. Dimethylsulfate footprint assays reveal a close interaction between TtgR and the central region of this operator. The results of analytical ultracentrifugation demonstrate that TtgR forms stable dimers in solution, and that two dimers bind to the operator. Microcalorimetric analysis of the binding of the two TtgR dimers to the cognate operator showed biphasic behavior, and an interaction model was developed for the cooperative binding of two TtgR dimers to their target operators. The binding of the two TtgR dimers to the operator was characterized by a Hill coefficient of 1.63+/-0.13 (k(D)=18.2(+/-6.3) microM, k(D)(')=0.91(+/-0.49) microM), indicating positive cooperativity. These data are in close agreement with the results of sedimentation equilibrium studies of TtgR-DNA complexes. A series of oligonucleotides were generated in which the imperfect palindrome of the TtgR operator was empirically optimized. Optimization of the palindrome did not significantly alter the binding of the initial TtgR dimer to the operator, but increased the cooperativity of binding and consequently the overall affinity. The minimal fragment for TtgR binding was a 30-mer DNA duplex, and analysis of its sequence revealed two partially overlapping inverted repeats co-existing within the large pseudo-palindrome operator. Based on the architecture of the operator, the thermodynamics of the process, and the TtgR-operator interactions we propose a model for the binding of TtgR to its target sequence.

Glyoxylate and pyruvate are antagonistic effectors of the Escherichia coli IclR transcriptional regulator.

The Escherichia coli isocitrate lyase regulator (IclR) regulates the expression of the glyoxylate bypass operon (aceBAK). Founding member of a large family of common fold transcriptional regulators, IclR comprises a DNA binding domain that interacts with the operator sequence and a C-terminal domain (C-IclR) that binds a hitherto unknown small molecule. We screened a chemical library of more than 150 metabolic scaffolds using a high-throughput protein stability assay to identify molecules that bind IclR and then tested the active compounds in in vitro assays of operator binding. Glyoxylate and pyruvate, identified by this method, bound the C-IclR domain with KD values of 0.9+/-0.2 and 156.2+/-7.9 microM, as defined by isothermal titration calorimetry. Both compounds altered IclR interactions with operator DNA in electrophoretic mobility shift assays but showed an antagonistic effect. Glyoxylate disrupted the formation of the IclR/operator complex in vitro by favoring the inactive dimeric state of the protein, whereas pyruvate increased the binding of IclR to the aceBAK promoter by stabilizing the active tetrameric form of the protein. Structures of the C-IclR domain alone and in complex with each effector were determined at 2.3 A, confirming the binding of both molecules in the effector recognition site previously characterized for the other representative of the family, the E. coli AllR regulator. Site-directed mutagenesis demonstrated the importance of hydrophobic patch formed by Met-146, Leu-154, Leu-220, and Leu-143 in interactions with effector molecules. In general, our strategy of combining chemical screens with functional assays and structural studies has uncovered two small molecules with antagonistic effects that regulate the IclR-dependent transcription of the aceBAK operon.

Ss-LrpB from Sulfolobus solfataricus condenses about 100 base pairs of its own operator DNA into globular nucleoprotein complexes.

Ss-LrpB from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2 is a member of the Lrp-like family of Bacterial/Archaeal transcription regulators that binds its own control region at three regularly spaced and partially conserved 15-bp-long imperfect palindromes. We have used atomic force microscopy to analyze the architecture of Ss-LrpB.DNA complexes with a different stoichiometry formed with the wild type operator and with an operator mutant. Binding of dimeric Ss-LrpB to all three target sites is accompanied by the formation of globular complexes, in which the protein induces strong DNA deformations. Furthermore, DNA contour length foreshortening of these complexes indicates DNA wrapping, with about 100 bp being condensed. The average bending angle is 260 degrees . The establishment of protein-protein contacts between Ss-LrpB dimers in these globular complexes will contribute to the cooperativity of the binding. The profound remodeling of the control region is expected to have a strong impact on gene expression and might constitute the key element in the autoregulatory process.

The manganese-responsive repressor Mur of Rhizobium leguminosarum is a member of the Fur-superfamily that recognizes an unusual operator sequence.

The manganese uptake regulator Mur of Rhizobium leguminosarum is a close homologue of the global iron regulatory protein Fur. Mur represses the sitABCD operon, which encodes a Mn2+ transport system, specifically in response to Mn2+ but not Fe2+. In previous work the authors mapped the 5' ends of two sit operon transcripts, termed TS1 and TS2, which were co-ordinately regulated by Mn2+-Mur, but this paper now shows that only TS1 is a primary transcript. DNase I protection analyses showed that purified Mur bound, with similar affinity, to two sites in the regulatory region of sitABCD, but only when Mn2+ was present in the reaction buffer. These Mn2+-Mur-binding sites, termed MRS1 and MRS2 (Mur-responsive sequence), were closely related in sequence to each other and were separated by 16 bp, spanning the transcription initiation site TS1. The extent of the protected DNA was 34 and 31 bp for MRS1 and MRS2, respectively, which is in accord with other members of the Fur family. The DNA sequences recognized by Mn2+-Mur are wholly different from conventional Fur boxes, but some similarities to a recognition sequence for the Fur regulator from Bradyrhizobium japonicum were noted. Transcription analysis of the R. leguminosarum mur gene showed its expression to be independent of Mn2+-Mur. Thus, Mur is a sequence-specific DNA-binding protein that responds in vitro to manganese, and thus can occlude RNA polymerase access to the sitABCD promoter. Moreover, Mur recognizes a DNA sequence atypical for the Fur superfamily and, like Fur from B. japonicum, defines a new subclass of Fur-like transcriptional regulators.

Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family.

The mechanisms by which Bacillus subtilis OhrR, a member of the MarR family of transcription regulators, binds the ohrA operator and is induced by oxidation of its lone cysteine residue by organic hydroperoxides to sulphenic acid are unknown. Here, we describe the crystal structures of reduced OhrR and an OhrR-ohrA operator complex. To bind DNA, OhrR employs a chimeric winged helix-turn-helix DNA binding motif, which is composed of extended eukaryotic-like wings, prokaryotic helix-turn-helix motifs, and helix-helix elements. The reactivity of the peroxide-sensing cysteine is not modulated by proximal basic residues but largely by the positive dipole of helix alpha1. Induction originates from the alleviation of intersubunit steric clash between the sulphenic acid moieties of the oxidized sensor cysteines and nearby tyrosines and methionines. The structure of the OhrR-ohrA operator complex reveals the DNA binding mechanism of the entire MarR family and suggests a common inducer binding pocket.

Integrative plasmid vector for constructing single-copy reporter systems to study gene regulation in Rhizobium meliloti and related species.

The integrative system of phage 16-3 of Rhizobium meliloti 41 was shown to function in several bacterial species belonging to the Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium genera. It might also function in many other bacterial species provided that both the target site (attB) and the required host factor(s) are present. Here we report on the construction of a new integrative vector that can be utilized in gene regulation studies. It provides an opportunity to create a single-copy set-up for characterizing DNA-protein interactions in vivo, in a wide range of bacteria. To demonstrate the usefulness of the vector, transcription repression by binding of the C repressor protein of phage 16-3 to wild type operators was studied. The assay system provided highly reproducible quantitative data on repression.

Improved model of a LexA repressor dimer bound to recA operator.

A complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Delta(1-67) S119A, S119A, G85D and Delta(1-67) quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the 'cleavable' cleavage site region found in the crystal structures is actually the more relevant form of the region in wild-type LexA since it agrees with the value of the pre-exponential Arrhenius factor for its autocleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a 'conformational switch' inferred from the crystal structures is meaningless.

Phenotypes of combined tet repressor mutants for effector and operator recognition and allostery.

Tet repressor mutants with a shifted effector specificity, preference for a mutant operator sequence or reversion of activity were combined to construct variants bearing two or three phenotypic alterations. TetR alleles with combinations of altered operator and effector specificities can be created by merging the respective residues in a single polypeptide. The mutations giving rise to revTetR, on the other hand, show drastic influences on the ligand binding phenotypes when combined with respective alterations. One TetR variant displays all three phenotypic alterations and thus demonstrates the general possibility of implementing them in one protein.

Hyperthermophilic Thermotoga arginine repressor binding to full-length cognate and heterologous arginine operators and to half-site targets.

The degree of sequence conservation of arginine repressor proteins (ArgR) and of the cognate operators (tandem pairs of 18 bp imperfect palindromes, ARG boxes) in evolutionarily distant bacteria is unusually high, and the global mechanism of ArgR-mediated regulation appears to be similar. However, here we demonstrate that the arginine repressor from the hyperthermophilic bacterium Thermotoga neapolitana (ArgR(Tn)) exhibits characteristics that clearly distinguish this regulator from the well-studied homologues from Escherichia coli, Bacillus subtilis and B.stearothermophilus. A high-resolution contact map of ArgR(Tn) binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgR(Tn) establishes all of its strong contacts with a single ARG box-like sequence of the operator only. Protein array and electrophoretic mobility-shift data demonstrate that ArgR(Tn) has a remarkable capacity to bind to arginine operators from Gram-negative and Gram-positive bacteria, and to single ARG box-bearing targets. Moreover, the overall effect of L-arginine on the apparent K(d) of ArgR(Tn) binding to various cognate and heterologous operator fragments was minor with respect to that observed with diverse bacterial arginine repressors. We demonstrate that this unusual behaviour for an ArgR protein can, to a large extent, be ascribed to the presence of a serine residue at position 107 of ArgR(Tn), instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor. Consistent with these results, ArR(Tn) was found to behave as a superrepressor in E.coli, inhibiting growth in minimal medium, even supplemented with arginine, whereas similar constructs bearing the S107Q mutant allele did not inhibit growth. We assume that ArgR(Tn), owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga

Arginine operator binding by heterologous and chimeric ArgR repressors from Escherichia coli and Bacillus stearothermophilus.

Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the alpha4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the alpha4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.

High resolution contact probing of the Lrp-like DNA-binding protein Ss-Lrp from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2.

Ss-Lrp, from Sulfolobus solfataricus, is an archaeal homologue of the global bacterial regulator Lrp (Leucine-responsive regulatory protein), which out of all genome-encoded proteins is most similar to Escherichia coli Lrp (E-value of 5.6 e-14). The recombinant protein has been purified as a 68 kDa homotetramer. The specific binding of Ss-Lrp to its own control region is suggestive of negative autoregulation. A high resolution contact map of Ss-Lrp binding was established by DNase I and hydroxyl radical footprinting, small non-intercalating groove-specific ligand-binding interference, and various base-specific premodification and base removal binding interference techniques. We show that Ss-Lrp binds one face of the DNA helix and establishes the most salient contacts with two major groove segments and the intervening minor groove, in a region that overlaps the TATA-box and BRE promoter elements. Therefore, Ss-Lrp most likely exerts autoregulation by preventing promoter recognition by TBP and TFB. Moreover, the results demonstrate profound Ss-Lrp induced structural alterations of sequence stretches flanking the core contact site, and reveal that the deformability of these regions significantly contributes to binding selectivity.

SmtB-DNA and protein-protein interactions in the formation of the cyanobacterial metallothionein repression complex: Zn2+ does not dissociate the protein-DNA complex in vitro.

The synechococcal metallothionein locus smt consists of two divergent genes: smtA coding for the metallothionein SmtA, and smtB coding for the trans-acting regulator SmtB. The latter binds at two inverted repeats, designated S1/S2 and S3/S4, in the overlapping promoter/operator sites between the two genes. We have determined the binding stoichiometries to the entire operator/promoter DNA and to the separate S1/S2 and S3/S4 half-operator oligonucleotides using sedimentation equilibrium and sedimentation velocity measurements. The full promoter/operator DNA binds two SmtB dimers. The hydrodynamic behavior of this complex supports a compact nucleoprotein structure. Each separate S1/S2 and S3/S4 operator sequence also binds two dimers. An equal molar mixture of separate S1/S2 and S3/S4 operator sequences, in excess SmtB, forms a S1/S2-SmtB:SmtB-S3/S4 bridge complex. Combining these results with previously published binding interference data, which showed consecutive S1/S2 and S3/S4 SmtB occupancy on the operator/promoter DNA, we have developed a model for the establishment of the repression complex that appears to involve significant DNA compaction, presumably DNA bending, stabilized by SmtB-SmtB bridge interactions. DNase I footprinting titrations also showed consecutive S1/S2 and S3/S4 SmtB occupancy. The footprints expand considerably in the presence of Zn2+. Hence, SmtB remains bound to the operator sites when Zn2+ ions are present. This result is further supported by gel retardation assay. Failure of the metal ions to dissociate SmtB from the DNA points to a hitherto unknown function of SmtB in the regulation of the smt locus.

The ratio between CcdA and CcdB modulates the transcriptional repression of the ccd poison-antidote system.

The ccd operon of the F plasmid encodes CcdB, a toxin targeting the essential gyrase of Escherichia coli, and CcdA, the unstable antidote that interacts with CcdB to neutralize its toxicity. Although work from our group and others has established that CcdA and CcdB are required for transcriptional repression of the operon, the underlying mechanism remains unclear. The results presented here indicate that, although CcdA is the DNA-binding element of the CcdA-CcdB complex, the stoichiometry of the two proteins determines whether or not the complex binds to the ccd operator-promoter region. Using electrophoretic mobility shift assays, we show that a (CcdA)2-(CcdB)2 complex binds DNA. The addition of extra CcdB to that protein-DNA complex completely abolishes DNA retardation. Based on these results, we propose a model in which the ratio between CcdA and CcdB regulates the repression state of the ccd operon. When the level of CcdA is superior or equal to that of CcdB, repression results. In contrast, derepression occurs when CcdB is in excess of CcdA. By ensuring an antidote-toxin ratio greater than one, this mechanism could prevent the harmful effect of CcdB in plasmid-containing bacteria.

Control of the arabinose regulon in Bacillus subtilis by AraR in vivo: crucial roles of operators, cooperativity, and DNA looping.

The proteins involved in the utilization of L-arabinose by Bacillus subtilis are encoded by the araABDLMNPQ-abfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene, and araR regulatory gene is induced by L-arabinose and negatively controlled by AraR. The purified AraR protein binds cooperatively to two in-phase operators within the araABDLMNPQ-abfA (OR(A1) and OR(A2)) and araE (OR(E1) and OR(E2)) promoters and noncooperatively to a single operator in the araR (OR(R3)) promoter region. Here, we have investigated how AraR controls transcription from the ara regulon in vivo. A deletion analysis of the ara promoters region showed that the five AraR binding sites are the key cis-acting regulatory elements of their corresponding genes. Furthermore, OR(E1)-OR(E2) and OR(R3) are auxiliary operators for the autoregulation of araR and the repression of araE, respectively. Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the ara operon requires communication between repressor molecules bound to two properly spaced operators. This communication implicates the formation of a small loop by the intervening DNA. In an in vitro transcription system, AraR alone sufficed to abolish transcription from the araABDLMNPQ-abfA operon and araE promoters, strongly suggesting that it is the major protein involved in the repression mechanism of L-arabinose-inducible expression in vivo. The ara regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control.