Purification of the Escherichia coli purine regulon repressor and identification of corepressors.

The Escherichia coli pur regulon repressor protein was overproduced in a phage T7 expression system. The overexpressed repressor constituted approximately 35% of the soluble cellular protein. Pur repressor was purified to near homogeneity by two chromatographic steps. Hypoxanthine or guanine was required for binding of purified repressor to purF operator DNA. Apparent dissociation constants of 3.4 nM were determined for binding of holorepressor to purF operator and of 1.7 and 7.1 microM were determined for aporepressor interaction with guanine and hypoxanthine, respectively. A requirement for hypoxanthine or guanine for conversion of aporepressor to holorepressor in vitro supports the earlier report (U. Houlberg and K.F. Jensen, J. Bacteriol. 153:837-845, 1983) that these purine bases are involved in regulation of pur gene expression in Salmonella typhimurium and confirms that hypoxanthine and guanine are corepressors.     

Regulation of the methionine regulon in Escherichia coli

The genes involved in methionine biosynthesis are scattered throughout the Escherichia coli chromosome and are controlled in a similar but not coordinated manner. The product of the metJ gene and S-adenosylmethionine are involved in the repression of this ‘regulon’.     

Structural characterization and corepressor binding of the Escherichia coli purine repressor.

The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine. We have characterized the purified repressor. Chemical cross-linking indicates that PurR is dimeric. Each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal 289-residue domain for corepressor binding. Each domain was isolated after cleavage by trypsin. Sites for dimer formation are present within the corepressor binding domain. The corepressors hypoxanthine and guanine bind cooperatively to distinct sites in each subunit. Competition experiments indicate that binding of one purine abolishes cooperativity and decreases the affinity and the binding of the second corepressor. Binding of each corepressor results in a conformation change in the corepressor binding domain that was detected by intrinsic fluorescence of three tryptophan residues. These experiments characterize PurR as a complex allosteric regulatory protein.     

Regulation of the Escherichia coli allantoin regulon: coordinated function of the repressor AllR and the activator AllS

The allantoin regulon of Escherichia coli, formed by three operons expressed from promoters allA(P), gcl(P) and allD(P), is involved in the anaerobic utilization of allantoin as nitrogen source. The expression of these operons is under the control of the repressor AllR. The hyperinduction of one of these promoters (allD(P)) by allantoin in an AllR defective mutant suggested the action of another regulator, presumably of activator type. In this work we have identified ybbS (proposed gene name allS), divergently transcribed from allA, as the gene encoding this activator. Analysis of the expression of the three structural operons in DeltaallS mutant showed that the expression from allD(P) was abolished, suggesting that AllS is essential for the expression of the corresponding operon. In a wild-type strain expression of allS takes place mainly anaerobically and is hyperinduced when the nitrogen source limits growth. However, expression of allS is independent of regulators of the Ntr response, NtrC or Nac. Band shift experiments showed that AllR binds to DNA containing the allS-allA intergenic region and the gcl(P) promoter and its binding is abolished by glyoxylate. Both DNA fragments contain a highly conserved inverted repeat, which after site-directed mutagenesis, has been proven to be the AllR-binding site. This site displays similarity with the IclR family recognized consensus. Interaction of AllR with the single operator present in the allS-allA intergenic region prevented binding of RNA polymerase to either of the two divergent promoters. The regulator AllS interacts only with allD(P) even in the absence of allantoin. Analysis of this promoter allowed us to identify an inverted repeat as a motif for AllS binding. We propose a model for the coordinate control of the allantoin regulon by AllR and AllS.     

Regulation of aroL expression by TyrR protein and Trp repressor in Escherichia coli K-12.

The promoter-operator region of the aroL gene of Escherichia coli K-12 contains three TYR R boxes and one TrpR binding site. Mutational analysis showed that TYR R boxes 1 and 3 are essential for TyrR-mediated regulation of aroL expression, while a fully functional TYR R box 2 does not appear to be essential for regulation. Regulation mediated by the TrpR protein required the TYR R boxes and TrpR site to be functional and was observed in vivo only with a tyrR+ strain. Under conditions favoring the formation of TyrR hexamers, DNase I protection experiments revealed the presence of phased hypersensitive sites, indicative of DNA backbone strain. This suggests that TyrR-mediated repression involves DNA looping. Purified TrpR protein protected the putative TrpR binding site in the presence of tryptophan, and this protection was slightly enhanced in the presence of TyrR protein. This result along with the in vivo findings implies that TyrR and TrpR are able to interact in some way. Inserting 4 bp between TYR R box 1 and the TrpR binding site results in increased tyrosine repression and the abolition of the tryptophan effect. Identification of a potential integration host factor binding site and repression studies of a himA mutant support the notion that integration host factor binding normally exerts a negative effect on tyrosine-mediated repression.     

Role of residue 147 in the gene regulatory function of the Escherichia coli purine repressor.

The crystal structures of corepressor-bound and free Escherichia coli purine repressor (PurR) have delineated the roles of several residues in corepressor binding and specificity and the intramolecular signal transduction (allosterism) of this LacI/GalR family member. From these structures, residue W147 was implicated as a key component of the allosteric response, but in many members of the LacI/GalR family, position 147 is occupied by an arginine. To understand the role of this tryptophan at position 147, three proteins, substituted by phenylalanine (W147F), alanine (W147A), or arginine (W147R), were constructed and characterized in vivo and in vitro, and their structures were determined. W147F displays a decreased affinity for corepressor and is a poor repressor in vivo. W147A and W147R, on the other hand, are super repressors and bind corepressor 13.6 and 7.9 times more tightly, respectively, than wild-type. Each mutant PurR-hypoxanthine-purF operator holo complex crystallizes isomorphously to wild-type. Whereas the apo corepressor binding domain (CBD) of W147F crystallizes under those conditions used for the wild-type protein, neither the apo CBD of W147R nor W147A crystallizes, although screened extensively for new crystal forms. Structures of the holo repressor mutants have been solved to resolutions between 2.5 and 2.9 A, and the structure of the apo CBD of W147F has been solved to 2.4 A resolution. These structures provide insight into the altered biochemical properties and physiological functions of these mutants, which appear to depend on the sometimes subtle preference for one conformation (apo vs holo) over the other.     

Overproduction of MalK protein prevents expression of the Escherichia coli mal regulon.

The mal regulon of Escherichia coli comprises a large family of genes whose function is the metabolism of linear maltooligosaccharides. Five gene products are required for the active accumulation of maltodextrins as large as maltoheptaose. Two cytoplasmic gene products are necessary and sufficient for the intracellular catabolism of these sugars. Two newly discovered enzymes have the capacity to metabolize these sugars but are not essential for their catabolism in wild-type cells. A single regulatory protein, MalT, positively regulates the expression of all of these genes in response to intracellular inducers, one of which has been identified as maltotriose. In the course of studying the mechanism of the transport system, we have placed the structural gene for one of the transport proteins, MalK, under the control of the Ptrc promoter to produce large amounts of this protein. We found that although high-level expression of MalK was not detrimental to E. coli, the increased amount of MalK decreased the basal-level expression of the mal regulon and prevented induction of the mal system even in the presence of external maltooligosaccharides. Constitutive mutants in which MalT does not depend on the presence of the internal inducer(s) were unaffected by the increased levels of the MalK protein. These results are consistent with the idea that MalK protein somehow interferes with the activity of the MalT protein. Different models for the regulatory function of MalK are discussed.     

Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon.

Two types of Escherichia coli K-12 regulatory mutants, partially or totally negative for the induction of the five catabolic enzymes (uronic isomerase, uxaC; altronate oxidized nicotinamide adenine dinucleotide: uxaB; mannonate hydrolyase, uxuA) and the transport system (exuT) of the hexuronate-inducible pathway, were isolated and analyzed enzymatically. Hexuronate-catabolizing revertants of the negative mutants showed a constitutive synthesis for some or all of these enzymes. Negative and constitutive mutations were localized in the same genetic locus, called exuR, and the following order for the markers situated between the min 65 and 68 was determined: argG--exuR--exuT--uxaC--uxaA--tolC. The enzymatic characterization of the pleiotropic negative and constitutive mutants of the exuR gene suggests that the exuR regulatory gene product exerts a specific and total control on the three exuT, uszB, and uxaC-uxaA operons of the galacturonate pathway and a partial control on the uxuA-uxuB operon of the glucuronate pathway. The analysis of diploid strains conatining both the wild type and a negative or constitutive allele of the exuR gene, as well as the analysis of thermosensitive mutants of the exuR gene, was in agreement with a negative regulatory mechanism for the control of the hexuronate system.     

Regulation of the multiple antibiotic resistance (mar) regulon by marORA sequences in Escherichia coli.

The mar operon and adjacent sequences were subcloned on a low-copy-number plasmid to identify essential regulatory elements. A 1.1-kbp fragment containing 133 bp of the operator-promoter region (marO), the full marRA gene sequences, and only 10 of 72 marB codons provided a dela mar strain with normal repressibility and inducibility and the ability to beget mar constitutive mutants.     

Regulation of components of the Pseudomonas aeruginosa phosphate-starvation-inducible regulon in Escherichia coli

Plasmids pPBP and pRS-XP containing the cloned genes for the Pseudomonas aeruginosa phosphate-starvation-inducible periplasmic phosphate-binding protein and outer membrane porin P (oprP), respectively, were introduced into various Escherichia coli Pho-regulon regulatory mutants. Using Western immunoblots and specific antisera, the production of both gene products was observed to be under the control of regulatory elements of the E. coli Pho regulon. Sequencing of the region upstream of the translational start site of the oprP gene revealed a 'Pho box' with strong homology to the E. coli consensus 'Pho box', the putative binding site of the PhoB activator. Since P. aeruginosa and E. coli belong to different families and have quite different GC contents, these data suggest strong evolutionary conservation of regulatory elements of the Pho regulon.     

Role of the catabolite activator protein in the maltose regulon of Escherichia coli.

The maltose regulon consists of three operons controlled by a positive regulatory gene, malT. Deletions of the gene crp were introduced into strains which carried a malT-lacZ hybrid gene. From the observed reduction in beta-galactosidase activity it was concluded that the expression of malT-lacZ, and therefore of malT, is controlled by the catabolite activator protein (CAP), the product of the gene crp. Mutations were obtained which allowed a malT-lacZ hybrid gene to be expressed at a high level even in the absence of CAP. These mutations were shown to be located in or close to the promoter of the malT gene and were called malTp. The malTp mutations were transferred in the cis position to a wild-type malT gene. In the resulting strains, the expression of two of the maltose operons, malEFG and malK-lamB, still required the action of CAP, whereas that of the third operon, malPQ, was CAP independent. Therefore, in wild-type cells, CAP appears to control malPQ expression mainly, if not solely, by regulating the concentration of MalT protein in the cell. On the other hand, it controls the other two operons more stringently, both by regulating malT expression and by a more direct action, probably exerted in the promoters of these operons.     

Crystallization and preliminary X-ray studies on the co-repressor binding domain of the Escherichia coli purine repressor.

The purine repressor is a putative helix-turn-helix DNA-binding protein that regulates several genetic loci important in purine and pyrimidine metabolism in Escherichia coli. The protein is composed of two domains, an N-terminal DNA-binding domain and a C-terminal core that binds the purine co-repressors, guanine and hypoxanthine. The co-repressor binding domain (residues 53 to 341) has been crystallized from polyethylene glycol 600-MgCl2 solutions. They are of the monoclinic form, space group P2(1), with a = 38.2 A, b = 125.7 A, c = 61.8 A and beta = 100.2 degrees. They diffract to a resolution of at least 2.2 A and contain two monomers per asymmetric unit. The importance of the structural determination of this domain is underscored by the high degree of sequence homology displayed within the effector binding sites among a sub-class of helix-turn-helix proteins, of which LacI and GalR are members. The structure of the PurR co-repressor binding domain will provide a high resolution view of one such domain and could serve as a possible model for future effector site structural determinations. Perhaps more important will be this structure's contribution to the further understanding of how protein-DNA interactions are modulated.     

大肠杆菌鼠李糖调节子

大肠杆菌（Escherichia coil）L-鼠李糖（rha）调节子由三个功能相关的操纵子（operon）组成,位于大肠杆菌染色体基因组中。它编码大肠杆菌吸收和利用L-鼠李糖的蛋白,即一个鼠李糖运输蛋白（RhaT）、三个鼠李糖代谢酶（RhaB、RhaA、RhaD）以及两个调节蛋白（RhaS、RhaR）。三个操纵子均受到L-鼠李糖本身的诱导,同时以调控蛋白RhaS、RhaR和CRP（cAMP受体蛋白）为中介的正调控也参与调节。