CytR Repressor Research Articles

A role for the interdomain linker region of the Escherichia coli CytR regulator in repression complex formation.

Regulatory complexes formed by the CytR repressor protein and the cAMP receptor protein (CRP) prevent transcription initiation from several promoters in Escherichia coli. The formation of the complexes is mediated by protein-DNA interactions and protein-protein interactions between the two regulators. Interestingly, co-binding with CRP has a profound effect on the configuration of the DNA-binding targets preferred by CytR. When binding to DNA by itself, CytR binds preferentially to two octamer repeats in direct or inverted orientation, and separated by 2 bp. However, in the presence of CRP, CytR recognizes inverted repeats separated by 10-13 bp, or direct repeats separated by 1 bp. A fixed orientation of at least one CytR octamer repeat in close proximity to a CRP-binding target is a common architectural feature at promoters optimised for repression complex formation. These observations suggest that CRP alters the DNA-binding mode of CytR. Here, we have investigated the CRP-induced changes in CytR by protein footprinting and alanine-scanning mutagenesis. Our data suggest that a flexible interdomain linker region in CytR, connecting the DNA-binding domain to the dimerization domain allows the repressor protein to interact with DNA-binding sites in a highly relaxed manner, as shown previously, and plays an active role in transcription regulation. Thus, the interactions between CRP, CytR and DNA within the repression complex appear to be more extensive than anticipated. The results support and extend the view that the high degree of adaptability observed in the CytR/CRP regulatory system is obtained though multiple adjustable interactions between the implicated factors.

Structural and Functional Analysis of the Promoter Region of the Escherichia coli udp Gene

Effect of mutations in the –10 and –35 regions of the udpgene promoter on the nature of its regulation by CytR and CRP proteins was studied. In studies of expression of mutant promoters, competition between RNA polymerase and the CytR repressor for the promoter region of the udp gene was shown. In the presence of the improved –10 region, the introduction of a substitution 15C → T (that is the presence of the elongated Pribnow block) resulted in the CRP-independent transcription of the udp gene promoter. The binding site CRP2 was shown to be indispensable for the maximum promoter activation by the transcription-activating cAMP–CRP complex. Both positive (cAMP–CRP complex) and negative (CytR) regulation of the promoter was virtually fully abolished after the introduction of mutations leading to the creation of canonical sequences in –10 and –35 promoter regions.

Functional interrelationship between elements of the Escherichia coli udp gene promotor responsible for binding regulatory proteins CytR, CRP, and RNA polymerase

Site-directed mutagenesis was conducted in the regulatory region of the Escherichia coli udp gene at promoter sites responsible for binding regulatory proteins CRP and CytR as well as RNA polymerase (the core-promoter containing the--10 sequence). In mutants with an "improved"--10 region, a partial relief from the control of the cAMP-CRP transcription activation complex occurred, and the negative CytR repressor regulation was reduced. In contrast, mutant promoters with a weak Pribnow block or with a deletion that completely eliminates the core-promoter exhibited an increased ability to titrate the CytR protein in vivo. On the other hand, the affinity of CytR for DNA in mutants with an altered--10 region was the same as in the wild-type udp promoter. After introduction of mutations affecting binding sites for CRP (CRP1 and CRP2), the negative effect of the CytR protein on promoter transcription was fully abolished. The CRP1 binding site was shown to play the main role in the activation of the promoter by the cAMP-CRP complex, whereas the CRP2 site participates in the formation of the repressor complex. Mutations in the main and additional CytR binding sites were isolated and characterized. On the basis of these data, it is concluded that the modification of each structural element of the udp regulatory region (binding sites for CytR, CRP, or RNA polymerase) caused changes in the overall pattern of the promoter regulation.

Role of Multiple CytR Binding Sites on Cooperativity, Competition, and Induction at the Escherichia coli udpPromoter

The CytR repressor fulfills dual roles as both a repressor of transcription from promoters of the Escherichia coli CytR regulon and a co-activator in some circumstances. Transcription is repressed by a three-protein complex (cAMP receptor protein (CRP)-CytR-CRP) that is stabilized by cooperative interactions between CRP and CytR. However, cooperativity also means that CytR can recruit CRP and, by doing so, can act as a co-activator. The central role of cooperativity in regulation is highlighted by the fact that binding of the inducer, cytidine, to CytR is coupled to CytR-CRP cooperativity; this underlies the mechanism for induction. Similar interactions at the different promoters of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism but also provide differential expression of these genes. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. Recently, we showed that CytR binds specifically to multiple sites in the E. coli deoP promoter, thereby providing competition for CRP binding to CRP operator site 1 (CRP1) and CRP2 as well as cooperativity. The effect of the competition at this promoter is to negate the role of CytR in recruiting CRP. Here, we have used quantitative footprint and mobility shift analysis to investigate CRP and CytR binding to the E. coli udp promoter. Here too, we find that CytR both cooperates and competes for CRP binding. However, consistent with both the distribution of CytR recognition motifs in the sequence of the promoter and the regulation of the promoter, the competition is limited to CRP2. When cytidine binds to CytR, the effect on cooperativity is very different at the udp promoter than at the deoP2 promoter. Cooperativity with CRP at CRP1 is nearly eliminated, but the effect on CytR-CRP2 cooperativity is negligible. These results are discussed in relation to the current structural model of CytR in which the core, inducer-binding domain is tethered to the helix-turn-helix, DNA-binding domain via flexible peptide linkers.

Protein-induced fit: the CRP activator protein changes sequence-specific DNA recognition by the CytR repressor, a highly flexible LacI member.

The CytR repressor and the cAMP receptor protein (CRP) bind cooperatively to several promoters in Escherichia coli to repress transcription initiation. The synergistic binding is mediated by protein-protein interactions between the two regulators. Here, in vitro selection experiments have been used to examine the DNA-binding characteristics of CytR, by itself and when co-binding with cAMP-CRP. We show that the optimal CytR-binding site consists of two octamer repeats, in direct or inverted orientation, and separated by 2 bp. However, when co-binding with cAMP-CRP, CytR instead recognizes inverted repeats separated by 10-13 bp, or direct repeats separated by 1 bp. The configurations of the latter set of operators correlate well with the configurations of natural CytR targets. Thus, cAMP-CRP induces conformational changes in CytR so that the repressor fits the natural targets. Most strikingly, CytR can adopt widely different conformations that are equally favored energetically for complex formation with cAMP-CRP. We propose that this structural adaptability is essential for CytR repression of promoters with diverse architectures. We discuss these novel concepts in the context of the CRP/CytR regulatory system, as well as the structural and functional implications for multiprotein-DNA complex formation in general.

Gene‐regulatory modules In Escherichia coli: nucleoprotein complexes formed by cAMP‐CRP and CytR at the nupG promoter

Repression by CytR depends on the formation of nucleoprotein complexes in which the CytR repressor and the cAMP-CRP activator complex bind co-operatively to the DNA. Transcription initiation from CytR-regulated promoters requires cAMP-CRP; therefore, the cAMP-CRP complex functions both as an activator and as a co-repressor in these promoters. Another interesting aspect of the CytR regulon is that each promoter appears to have individual features. Therefore, structural and functional rules governing the formation of repression and activation complexes in one promoter may not be valid for other promoters of the CytR regulon. Here we show that the Escherichia coli nupG gene contains one CytR- and four CRP-binding sites in the control region. Notably, the architecture of the CytR binding site is different from previously described targets. In addition, the CytR repressor triggers a DNA repositioning of a cAMP-CRP complex in the -35 region upon binding to its operator. Thus, formation of the repression and activation complexes at the nupG promoter involves different subsets of CRP-binding sites. These findings show that the bacterium uses positive and negative regulatory modules to differentially control the expression of CytR- and cAMP-CRP-regulated genes.

The gene encoding the periplasmic cyclophilin homologue, PPlase A, in Escherichia coli, is expressed from four promoters, three of which are activated by the cAMP–CRP complex and negatively regulated by the CytR repressor

The rot gene in Escherichia coli encodes PPIase A, a periplasmic peptidyl-prolyl cis-trans isomerase with homology to the cyclophilin family of proteins. Here it is demonstrated that rot is expressed in a complex manner from four overlapping promoters and that the rot regulatory region is unusually compact, containing a close array of sites for DNA-binding proteins. The three most upstream rot promoters are activated by the global gene regulatory cAMP-CRP complex and negatively regulated by the CytR repressor protein. Activation of these three promoters occurs by binding of cAMP-CRP to two sites separated by 53 bp. Moreover, one of the cAMP-CRP complexes is involved in the activation of both a Class I and a Class II promoter. Repression takes place by the formation of a CytR/cAMP-CRP/DNA nucleoprotein complex consisting of the two cAMP-CRP molecules and CytR bound in between. The two regulators bind co-operatively to the DNA overlapping the three upstream promoters, simultaneously quenching the cAMP-CRP activator function. These results expand the CytR regulon to include a gene whose product has no known function in ribo- and deoxyribonucleoside catabolism or transport.

Protein-protein interactions in gene regulation: The cAMP-CRP complex sets the specificity of a second DNA-binding protein, the CytR repressor

Maximal repression by the CytR protein depends on the formation of nucleoprotein complexes in which CytR interacts with DNA and with cAMP-cAMP receptor protein (CRP). Here we demonstrate that CytR regulates transcription from deoP2 promoters in which the entire CytR recognition sequence has been eliminated. Furthermore, CytR proteins deleted for the DNA-binding domain repress deoP2 in vivo and interact with deoP2 in vitro in a strictly cAMP-CRP-dependent fashion. These experiments show that the site of action of CytR can be specified by protein-protein interactions to cAMP-CRP, whereas CytR-DNA interactions may primarily serve to stabilize the nucleoprotein complex. This type of specificity mechanism may represent a general concept in the recruitment of DNA-binding proteins in combinatorial regulatory systems.

Characterization of cytR mutations that influence oligomerization of mutant repressor subunits.

In Escherichia coli, the transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. The role of the CytR repressor in transcriptional regulation has been examined through a study of mutant CytR proteins. Two important and interrelated CytR mutants are encoded by cytR delta M149, a dominant negative allele, and cytRC289R. Studies with CytR delta M149 indicated that the native, repression-competent CytR protein is multimeric while the CytR amino acid substitution C-289-->R has been proposed to affect subunit oligomerization on the basis of its ability to suppress the transdominance of CytR delta M149. The present study identifies other CytR amino acid residues proximal to Cys-289 that may also participate in normal subunit oligomerization. Mutations in these CytR residues, cytRA307P, cytRM308R, and cytRL309P, encoded inactive repressors in a CytR- background and, when combined with cytR delta M149, yielded hybrid repressors that were recessive in a CytR+ genetic background. Because the stability and solubility observed for the new, mutant CytR proteins and the wild-type CytR protein were indistinguishable, these residue replacements, like the C-289-->R substitution, are envisaged as being located at the subunit interface and thus suppress the CytR delta M149 transdominance by blocking efficient and stable assembly of wild-type and hybrid CytR subunits. The assignment of CytR amino acids to a protein region involved in subunit association is also consistent with the observations that these CytR amino acids are roughly colinear with regions of the LacI repressor that influence monomer-dimer association and would be surface located by alignment to the E. coli galactose-binding protein crystal structure.

Identification of the nucleotide sequence recognized by the cAMP-CRP dependent CytR repressor protein in the deoP2 promoter in E. coli.

In E. coli repression of transcription initiation by the CytR protein relies on CytR-DNA interactions as well as on interactions between CytR and the cAMP-CRP activator complex. To identify the nucleotide sequence recognized by CytR, mutants of the deoP2 promoter with a reduced regulatory response to CytR have been isolated. Five single bp mutation derivatives of deoP2 with a 2-5-fold decrease in CytR regulation have been characterized. In vitro, the only effect of the mutations was a decrease in the binding affinity of CytR, and a clear correlation was observed between the reduction in CytR regulation in vivo and the reduction in CytR binding in vitro. The mutations all reside in a sequence element that contains an imperfect direct as well as an imperfect inverted repeat. As the active form of CytR, most likely, is an oligomer with two-fold rotational symmetry, CytR probably interacts with the inverted repeat. Degenerate versions of the inverted repeat are present in all CytR binding sites characterized so far, however, the distance between the half-sites varies.

CAMP-CRP activator complex and the CytR repressor protein bind co-operatively to the cytRP promoter in Escherichia coli and CytR antagonizes the cAMP-CRP-induced DNA bend

Initiation of transcription from the cytRP promoter in Escherichia coli is activated by the cAMP-CRP complex and negatively regulated by the CytR repressor protein. By combining gel retardation and footprinting assays, we show that cAMP-CRP binds to a single site centered at position −64 and induces a considerable bend in the DNA. CytR binds to a region immediately downstream from, and partially overlapping, the CRP site, and induces a modest bend into the DNA. In combination, cAMP-CRP and CytR bind co-operatively to cytRP forming a nucleoprotein complex in which the proteins directly interact with each other and bind to the same face of the DNA helix. CytR binding concomitantly antagonizes the cAMP-CRP-induced bend. This study indicates that the minimal DNA region required to obtain CytR regulation consists of a single binding site for each of cAMP-CRP and CytR. The case described here, in which a protein-induced DNA bend is modulated by a second protein, may illustrate a mechanism that applies to other regulatory systems.

Restored DNA-binding of the cAMP-CRP activator complex reestablishes negative regulation by the CytR repressor in the deoP2 promoter in Escherichia coli.

We have investigated in vivo the coupling between CytR regulation of the deoP2 promoter in Escherichia coli and the DNA-binding specificity of the cAMP-CRP (cAMP receptor protein) complex in order to obtain a more detailed picture of the role played by cAMP-CRP in CytR regulation. By introducing CRP proteins that exhibit an altered DNA binding specificity into a strain containing a mutant deoP2 promoter in which cAMP-CRP activation was decreased and CytR regulation completely abolished, we show that CytR regulation of this promoter can be reestablished by restored the DNA binding of the cAMP-CRP complex. Hence, CytR regulation of deoP2 can be modulated by simply varying DNA binding of cAMP-CRP. These data confirm the crucial role played by the cAMP-CRP activator complex in CytR regulation of the deoP2 promoter.

The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12

The tsx-p2 promoter is one of at least seven Escherichia coli promoters that are activated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and negatively regulated by the CytR repressor. DNase I footprinting assays were used to study the interactions of these regulatory proteins with the tsx-p2 promoter region and to characterize tsx-p2 regulatory mutants exhibiting an altered response to CytR. We show that the cAMP-CRP activator complex recognizes two sites in tsx-p2 that are separated by 33 bp: a high-affinity site (CRP-1) overlaps the -35 region, and a low-affinity site (CRP-2) is centered around position -74 bp. The CytR repressor protects a DNA segment that is located between the two CRP sites and partially overlaps the CRP-1 target. In combination, the cAMP-CRP and CytR proteins bind cooperatively to tsx-p2, and the nucleoprotein complex formed covers a region of 78 bp extending from the CRP-2 site close to the -10 region. The inducer for the CytR repressor, cytidine, does not prevent in vitro DNA binding of CytR, but releases the repressor from the nucleoprotein complex and leaves the cAMP-CRP activator bound to its two DNA targets. Thus, cytidine interferes with the cooperative DNA binding of cAMP-CRP and CytR to tsx-p2. We characterized four tsx-p2 mutants exhibiting a reduced response to CytR; three carried mutations in the CRP-2 site, and one carried a mutation in the region between CRP-1 and the -10 sequence. Formation of the cAMP-CRP-CytR DNA nucleoprotein complex in vitro was perturbed in each mutant. These data indicate that the CytR repressor relies on the presence of the cAMP-CRP activator complex to regulate tsx-p2 promoter activity and that the formation of an active repression complex requires the combined interactions of cAMP-CRP and CytR at tsx-p2.

Heterologous cooperativity in Escherichia coli. The CytR repressor both contacts DNA and the cAMP receptor protein when binding to the deoP2 promoter.

Promoters in Escherichia coli that are negatively regulated by the CytR repressor are also activated by the cAMP receptor protein (CRP) complexed to cAMP; as a characteristic, these promoters encode two binding sites for the cAMP.CRP complex. Biochemical and genetic studies have shown that CytR relies on interactions with the cAMP.CRP complex in order to bind promoter DNA and repress transcription. Here we have purified CytR to near homogeneity and addressed the question of how it interacts with the deoP2 promoter. Gel retardation and DNase I footprinting analyses show that CytR is a true sequence-specific DNA-binding protein that binds to the sequence between the two CRP sites in deoP2 with a relatively low affinity. In the presence of the cAMP.CRP complex the two protein species bind cooperatively to deoP2, forming a complex in which CytR occupies the sequence between the two DNA bound cAMP.CRP complexes. Furthermore, the inducer (cytidine) does not affect independent DNA binding of CytR, rather the CytR/cAMP.CRP cooperativity is perturbed. These results indicate that CytR binding to deoP2 relies on both repressor-DNA interactions and protein-protein interactions to cAMP.CRP. This combinatorial repression mechanism, in which an activator functions as an adaptor for a repressor that is not capable of blocking transcription on its own, is unprecedented in prokaryotes; it is, however, reminiscent of repression mechanisms found in eukaryotes.

The malX malY operon of Escherichia coli encodes a novel enzyme II of the phosphotransferase system recognizing glucose and maltose and an enzyme abolishing the endogenous induction of the maltose system

Mutants lacking MalK, a subunit of the binding protein-dependent maltose-maltodextrin transport system, constitutively express the maltose genes. A second site mutation in malI abolishes the constitutive expression. The malI gene (at 36 min on the linkage map) codes for a typical repressor protein that is homologous to the Escherichia coli LacI, GalR, or CytR repressor (J. Reidl, K. Römisch, M. Ehrmann, and W. Boos, J. Bacteriol. 171:4888-4899, 1989). We now report that MalI regulates an adjacent and divergently oriented operon containing malX and malY. MalX encodes a protein with a molecular weight of 56,654, and the deduced amino acid sequence of MalX exhibits 34.9% identity to the enzyme II of the phosphototransferase system for glucose (ptsG) and 32.1% identity to the enzyme II for N-acetylglucosamine (nagE). When constitutively expressed, malX can complement a ptsG ptsM double mutant for growth on glucose. Also, a delta malE malT(Con) strain that is unable to grow on maltose due to its maltose transport defect becomes Mal+ after introduction of malI::Tn10 and the plasmid carrying malX. MalX-mediated transport of glucose and maltose is likely to occur by facilitated diffusion. We conclude that malX encodes a phosphotransferase system enzyme II that can recognize glucose and maltose as substrates even though these sugars may not represent the natural substrates of the system. The second gene in the operon, malY, encodes a protein of 43,500 daltons. Its deduced amino acid sequence exhibits weak homology to aminotransferase sequences. The presence of plasmid-encoded MalX alone was sufficient for complementing growth on glucose in a ptsM ptsG glk mutant, and the plasmid-encoded MalY alone was sufficient to abolish the constitutivity of the mal genes in a malK mutant. The overexpression of malY in a strain that is wild type with respect to the maltose genes strongly interferes with growth on maltose. This is not the case in a malT(Con) strain that expresses the mal genes constitutively. We conclude that malY encodes an enzyme that degrades the inducer of the maltose system or prevents its synthesis.

Single amino acid substitutions in the cAMP receptor protein specifically abolish regulation by the CytR repressor in Escherichia coli.

Promoters in Escherichia coli that are negatively regulated by the CytR repressor are also activated by the cAMP receptor protein (CRP) complexed to cAMP; as a characteristic, these promoters encode tandem binding sites for cAMP-CRP. In one such promoter, deoP2, CytR binds to the region between the tandem CRP binding sites with a relatively low affinity; in the presence of cAMP-CRP, however, the repressor and activator bind cooperatively to the DNA. Here we have investigated this cooperativity by isolating mutants of the CRP protein that abolish CytR regulation without exhibiting a concomitant loss in their ability to activate transcription. Four different, single amino acid substitutions in CRP give rise to this phenotype. These amino acids lie in close proximity on the surface of the CRP tertiary structure in a portion of the protein that is not in contact with the DNA. In vitro analyses of one of the CRP mutants show that it interacts with the DNA in a manner indistinguishable from wild-type CRP, whereas its interaction with CytR is perturbed. These results strongly indicate that cooperative DNA binding of CytR and cAMP-CRP is achieved through protein-protein interactions.

A novel function of the cAMP-CRP complex in Escherichia coli: cAMP-CRP functions as an adaptor for the CytR repressor in the deo operon.

Unlike classical bacterial repressors, the CytR repressor of Escherichia coli cannot independently regulate gene expression. Here we show that CytR binding to the deoP2 promoter relies on interaction with the master gene regulatory protein, CRP, and, furthermore, that cAMP-CRP and CytR bind co-operatively to deoP2. Using mutant promoters we show that tandem, properly spaced DNA-bound cAMP-CRP complexes are required for this co-operative binding. These data suggest that CytR forms a bridge between tandem cAMP-CRP complexes, and that cAMP-CRP functions as an adaptor for CytR. The implications of this new version of negative control in E. coli on bacterial gene expression and on combinatorial gene regulation in higher organisms are discussed.

The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) CytR selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the cAMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR. These findings indicate that both CRP targets are required for efficient CytR repression of deoCp2. Models for the action of CytR are discussed in light of these findings.

Tandem DNA-bound cAMP-CRP complexes are required for transcriptional repression of the deoP2 promoter by the CytR repressor in Escherichia coli.

We have studied the deoP2 promoter in Escherichia coli to define features important for its interaction with the CytR repressor. As is characteristic for CytR-regulated promoters, deoP2 encodes tandem binding sites for the activating complex cAMP-CRP. One of these sites, CRP-1, overlaps the - 35 region, and is sufficient for activation; the second site, CRP-2, centred around-93, is indispensable for repression. Here we demonstrate, by means of in vivo titration, that CytR interaction with deoP2 depends not only on CRP-2, but also on CRP-1 and the length and possibly the sequence separating these two sites. Also, point mutations in either CRP site reduce or abolish CytR titration; however, no co-operativity is observed in the interaction of CytR with the two CRP binding sites. Furthermore, the reduction in CytR titration parallels the reduction in binding of cAMP-CRP to the mutated CRP sites in vitro. These observations are not easily explained by current models for the action of prokaryotic repressors; instead we favour a model in which the interaction of CytR with deoP2 depends on the presence of tandem DNA-bound cAMP-CRP complexes.

Analysis of the regulatory region of the Escherichia coli nupG gene, encoding a nucleoside‐transport protein

The S1-mapping procedure, when applied to an 800-bp fragment upstream of the nupG structural gene of Escherichia coli revealed one transcription-initiation site. The corresponding promoter is negatively regulated by the cytR and the deoR repressors. The expression of the gene is activated by the complex between cAMP and its receptor protein. In strains lacking cAMP the promoter expression is reduced and it is no longer regulated by the cytR repressor, whereas the deoR regulation is retained.