ARG Boxes Research Articles

Osmoregulation of dimer resolution at the plasmid pJHCMW1 mwr locus by Escherichia coli XerCD recombination.

Xer-mediated dimer resolution at the mwr site of plasmid pJHCMW1 is osmoregulated in Escherichia coli. Whereas under low-salt conditions, the site-specific recombination reaction is efficient, under high-salt conditions, it proceeds inefficiently. Regulation of dimer resolution is independent of H-NS and is mediated by changes in osmolarity rather than ionic effects. The low level of recombination at high salt concentrations can be overcome by high levels of PepA or by mutating the ARG box to a sequence closer to the E. coli ARG box consensus. The central region of the mwr core recombination site plays a role in regulation of site-specific recombination by the osmotic pressure of the medium.

Transcription regulation in thermophilic Bacteria: high resolution contact probing of Bacillus stearothermophilus and Thermotoga neapolitana arginine repressor-operator interactions

Arginine-mediated regulation is remarkably well conserved in very divergent bacteria, and shows a number of unusual features that distinguish arginine regulation from other transcriptional control mechanisms. The arginine repressor subunit consists of a basic N-terminal DNA-binding domain, which belongs to the winged helix-turn-helix family, connected through a flexible linker to an acidic C-terminal domain responsible for binding of arginine and assembly of the high-affinity holohexamer, which binds an approximately 40 bp target. To gain further insight into the molecular details of arginine repressor-operator interactions we have established a high resolution contact map of the argC operator from Bacillus stearothermophilus, a moderate thermophilic Gram-positive bacterium, and the argR operator from Thermotoga neapolitana, a Gram-negative hyperthermophile, with the corresponding ArgR proteins. Enzymatic and chemical footprinting have been combined with missing contact, pre-modification, base substitution, and small ligand binding interference techniques to gather information on backbone and base-specific contacts with major and minor groove determinants of the operators. Wild-type and mutant argC operators have been compared for their interaction with the repressor, using both in vivo and in vitro approaches. Our results indicate that the operators of B. stearothermophilus and T. neapolitana consist of two ARG box-like sequences, 18 bp imperfect palindromes, separated by two and three base-pairs, respectively, and that the repressors from thermophilic origin establish base-specific contacts with two major groove segments and the intervening minor groove of each ARG box, all aligned on one face of the helix. In contrast, no specific contacts are established in the minor groove facing the repressor in the centre of the operator, nevertheless this region plays a crucial structural role in complex formation, as indicated by mutant studies. This picture is reminiscent of arginine repressor binding in Escherichia coli, and therefore reinforces the uniform view of arginine regulation, but also reveals a number of striking differences at particular positions of the boxes and in the length and base-pair composition of the spacer connecting two ARG boxes in the operator. These might be responsible, in part, for subtle but important functional and mechanistic differences in the way species-specific repressors interact wth their cognate target sites. These variations are underlined by the different behaviour of the repressors from E. coli, B. stearothermophilus and T. neapolitana in their potential to bind heterologous operators, their requirement for arginine, and the resistance of complex formation to non-specific competitor DNA. Our findings are discussed in view of the crystal structure of the arginine repressor from B. stearothermophilus.

Conservation of the binding site for the arginine repressor in all bacterial lineages.

The arginine repressor ArgR/AhrC is a transcription factor universally conserved in bacterial genomes. Its recognition signal (the ARG box), a weak palindrome, is also conserved between genomes, despite a very low degree of similarity between individual sites within a genome. Thus, the arginine repressor is different from two other universal transcription factors - HrcA, whose recognition signal is very strongly conserved both within and between genomes, and LexA/DinR, whose signal is strongly conserved within, but not between, genomes. The arginine regulon is well studied in Escherichia coli and to some extent in Bacillus subtilis and some other genomes. Here, we apply the comparative genomic approach to the prediction of the ArgR-binding sites in all completely sequenced bacterial genomes. Orthologs of ArgR/AhrC were identified in the complete genomes of E. coli, Haemophilus influenzae, Vibrio choleras, B. subtilis, Mycobacterium tuberculosis, Thermotoga maritima, Chlamydia pneumoniae and Deinococcus radiodurans. Candidate arginine repressor binding sites were identified upstream of arginine transport and metabolism genes. We found that the ArgR/AhrC recognition signal is conserved in all genomes that contain genes encoding orthologous transcription factors of this family. All genomes studied except M. tuberculosis contain ABC transport cassettes (related to the Art system of E. coli) belonging to the candidate arginine regulons.

Probing Activation of the Prokaryotic Arginine Transcriptional Regulator Using Chimeric Proteins

The major transcription factors controlling arginine metabolism in Escherichia coli andBacillus subtilis, ArgR and AhrC, respectively, are homologous multimeric proteins that form l -arginine-dependent DNA-binding complexes capable of repressing transcription of the biosynthetic genes (both), activating transcription of catabolic genes (AhrC only) or facilitating plasmid dimer resolution (both). Multimerisation and l -arginine binding are associated with the C-terminal 70-80 residues; the N-terminal regions contain a winged helix-turn-helix DNA-binding domain. We have constructed chimeric genes in which the sequences for the N and C-terminal domains have been swapped. The resultant chimeric proteins and their corresponding native proteins have been analysed for their ability to multimerise and bind DNA operator sites in an l -arginine-dependent fashion.Gel filtration and equilibrium sedimentation analysis are consistent with the formation of hexamers by all four proteins in the presence of l -arginine and at high protein concentrations (>100nM monomer). The hexamer sedimentation coefficients suggest that there is a reduction in molecular volume upon binding L -arginine, consistent with a conformational change accompanying an allosteric activation of DNA-binding. In the absence of l -arginine or at lower protein concentrations, the hexamers are clearly in rapid equilibrium with smaller subunits, whose dominant species appear to be based on trimers, as expected from the crystal structure of the ArgR C-terminal fragment, with the exception of the ArgR-C chimera, which apparently dissociates into dimers, suggesting that in the intact protein the DNA-binding domains may have a significant dimeric interaction. The hexamer-trimer Kdis in the micromolar range, suggesting that trimers are the principal species at in vivo concentrations.DNA binding by all four proteins has been probed by gel retardation and DNase I footprinting analysis using all three types of naturally occurring operators: biosynthetic sites encompassing two 18bp ARG boxes separated by 2bp; biosynthetic sites containing two such boxes and a third 18bp ARG box at a distance of 100bp downstream, i.e. within the structural gene; and finally a catabolic operator which contains a single ARG box site. The data show that all four proteins bind to the operators at the expected regions in an l -arginine-dependent fashion. From the apparent affinities of the chimeras for each target site, there is no obvious sequence-specificity associated with the N-terminal domains; rather the data can be interpreted in terms of differential allosteric activation, including DNA binding in the absence ofl -arginine.Remarkably, the proteins show apparent “anti-competition” in the presence of excess, specific DNA fragments in gel retardation. This appears to be due to assembly of an activated form of the protein, probably hexamers, on the operator DNA. The data are discussed in terms of the current models for the mode of action of both native proteins.

The arcABDC gene cluster, encoding the arginine deiminase pathway of Bacillus licheniformis, and its activation by the arginine repressor argR.

The arginine deiminase pathway enables Bacillus licheniformis to grow anaerobically on arginine. Both the presence of arginine and anaerobiosis are needed to trigger induction of the pathway. In this study we have cloned and sequenced the arc genes encoding the pathway. They appear clustered in an operon-like structure in the order arcA (arginine deiminase), arcB (ornithine carbamoyltransferase), arcD (putative arginine-ornithine antiporter), arcC (carbamate kinase). It was found that B. licheniformis has an arginine repressor, ArgR, homologous to the B. subtilis arginine repressor AhrC. Mutants affected in argR were isolated. These mutants have lost both repression by arginine of the anabolic ornithine carbamoyltransferase and induction of the arginine deiminase pathway. Electrophoretic band shift experiments and DNase I footprinting revealed that in the presence of arginine, ArgR binds to a site upstream from the arc promoter. The binding site is centered 108 nucleotides upstream from the transcription start point and contains a single Arg box.

The arginine repressor of Escherichia coli K-12 makes direct contacts to minor and major groove determinants of the operators

In order to gain further insight into the molecular mechanism of arginine-dependent operator recognition by the hexameric Escherichia coli arginine repressor we have probed protein-DNA interactions in vitro and in vivo. We have extensively applied the chemical modification-protection and premodification-interference approach to two operators, the natural operator overlapping the P2 promoter of the carAB operon and a fully symmetrical consensus sequence. Backbone contacts were revealed by hydroxyl radical footprinting and phosphate ethylation interference. Base-specific contacts to purines and pyrimidines were revealed by methylation protection and premodification interference, KMnO4 and NH2OH.HCl-specific modification of thymine and cytosine residues, base-removal (depurination and depyrimidation), and base substitution (uracil and inosine). Additional information on the groove specificity of repressor binding was obtained by small ligand binding interference (distamycin and methyl green). In vivo, we measured the effects on the repressibility of 24 single base-pair substitutions obtained by saturation mutagenesis of half an Arg box in the carAB operator. The results of these experiments point to the conclusion that a hexameric arginine repressor molecule covers four turns of the helix, makes base-specific contacts to at least one guanine (G4 or G4′) and two thymine (T3, T13′, or T3′, T13) residues in each one of four consecutive major grooves on one face of the helix and with four A-T/T-A base-pairs, comprising the adenine residues A9, 9′, 12, 12′ and the thymine residues T10, 10′, 11, 11′, in the two outermost minor grooves of the operator, on the very same face of the DNA molecule. The hydrophobic 5-methyl groups of four thymine residues (T3, 3′, 13, 13′) in each Arg box contribute to major groove-specific recognition via hydrophobic and/or van der Waals interactions. The importance of minor groove contacts was further supported by the drastic effect of distamycin binding interference. In vivo, the most pronounced drops in repressibility were occasioned by mutations at positions 10 (A → G or C), 11 (T → A or G) and 12 (A → G, T or C).

Operator interactions by the Bacillus subtilis arginine repressor/activator, AhrC: novel positioning and DNA-mediated assembly of a transcriptional activator at catabolic sites.

We have previously reported the initial characterization of a catabolic operator site (O[rocA]) for the Bacillus subtilis arginine repressor/activator protein AhrC. Here, we present the characterization by gel retardation and DNase I footprinting of both O(rocA) and a second catabolic operator site, O(rocD). Both operator sites encompass a single recognition site, an ARG box, located immediately upstream of the transcriptional start points, a unique positioning for a transcriptional activator protein. Although there is considerable sequence homology between the two catabolic operator sites, they vary significantly, around twofold, in their apparent affinities for the protein (K'd approximately 90 nM for O[rocA] and approximtaely 190nM for O[rocD]). This difference may result from the lower match to the ARG box consensus of the O(rocD) site. Both catabolic operators show evidence for co-operative binding with respect to protein concentration. Determination of the sequences of two AhrC catabolic operator sites, in combination with the three such biosynthetic sites, has allowed the derivation of an improved B. subtilis ARG box consensus sequence, CATGAATAAAAATg/tCAAg/t. This is not identical to the Escherichia coli consensus operator for the AhrC homologue, ArgR, which may explain the only partial cross-functioning of these proteins in vivo. The O(rocA) site is adjacent to a sharp, stable bend located 5' to the catabolic operator. Circular permutation analysis has been used to determine the relative angle of bend (approximately 50 degrees), its location and the effect of adding magnesium ions and/or AhrC protein. Protein binding increases the relative bend angle to approximately 85 degrees. Bending is shown to be associated with a number of A-tracts in the upstream sequence. However, altering the phasing of the A-tracts has little effect on the affinity for AhrC. Truncation and competition experiments have been used to investigate the possible role of sequences flanking the operator on affinity. Very surprisingly, the affinity of the O(rocA) site appears to increase in the presence of excess, specific competitor fragment, i.e. the system shows anti-competitive effects. Competition is restored at high molar excesses of specific fragment over the protein. We propose a novel model for the assembly of a higher affinity form of AhrC at operator sites that is consistent with both the apparent co-operativity of binding and the anti-competitive effects. These data suggest that the molecular interactions occurring between the prokaryotic arginine-regulatory proteins and their operators may be more complex than is generally appreciated.

DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state.

The Escherichia coli arginine repressor (ArgR) controls expression of the arginine biosynthetic genes and acts as an accessory protein in Xer site-specific recombination at cer and related plasmid recombination sites. The hexameric wild-type protein shows L-arginine-dependent DNA binding. In this work, ArgR mutants that are defective in trimer-trimer interactions and bind DNA as trimers in an L-arginine-independent manner are isolated and characterized. Whereas the wild-type ArgR hexamer exhibits high-affinity binding to two repeated ARG boxes separated by 3 bp (each ARG box containing two identical dyad symmetrical 9 bp half-sites), the trimeric mutants bind to and footprint three adjacent half-sites of this 'idealized' substrate. Trimeric ArgR is impaired in its ability to repress the arginine biosynthetic genes and in Xer site-specific recombination. In the absence of L-arginine, residual wild-type ArgR-binding occurs as trimers. The binding of an N-terminal 77-amino-acid DNA-binding domain to idealized ARG boxes is also characterized.

Arginine biosynthesis and regulation in Lactobacillus plantarum: the carA gene and the argCJBDF cluster are divergently transcribed.

A cluster of citrulline biosynthetic genes has been cloned and sequenced from a fragment of Lactobacillus plantarum CCM 1904 (ATCC 8014) DNA isolated as complementing a Bacillus subtilis argF mutation. The gene order was carA-argCJBDF, with carA transcribed divergently from the arg cluster. Although other gram-positive bacteria show similar arg clusters, this arrangement for carA is thus far unprecedented. Downstream from the arg cluster, two open reading frames (ORF7 and ORF8) having unknown functions were found. Sequence analysis of the end of a 10.5-kb cloned DNA fragment showed that argF was 3.5 kb from the ldhL gene coding for L-(+)-lactate dehydrogenase. A tree representation of amino acid sequence clustering relationships of 31 ornithine carbamoyltransferases (OTCases) from various organisms revealed two prokaryotic groups: one with ArgF of L. plantarum and one with ArgF of B. subtilis, which are paralogous. This divergence was not observed in vivo because an L. plantarum argF mutant (AM 1215) harboring no OTCase activity was complemented by the argF genes of L. plantarum and B. subtilis. No OTCase activity was detectable when L. plantarum was grown in the presence of saturating amounts of arginine or citrulline. Arginine may repress the citrulline biosynthetic genes in L. plantarum by using 11 identified DNA motifs which resemble the Escherichia coli ARG box consensus and which are in most cases separated by multiples of 11 bp, corresponding to a DNA helical turn. The carA and argCJBDF genes are divergently transcribed. Their putative promoters are 6 bp apart and are partially overlapped by putative ARG boxes, suggesting concerted transcription regulation.

Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains.

Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.

Explanation for different types of regulation of arginine biosynthesis in Escherichia coli B and Escherichia coli K12 caused by a difference between their arginine repressors

In Escherichia coli K12, formation of the enzymes of arginine biosynthesis are controlled by arginine, with complete repression during growth with added arginine, severe repression (about 95%) during growth without added arginine and complete derepression during arginine-limited growth. In E. coli B, the degree of repression is not correlated with arginine concentrations. Under all conditions of growth enzyme formation is repressed, with repression being somewhat less in a medium with arginine than in a medium without arginine. These differences in repressibility between the two strains have been shown previously to be due to the presence of different alleles of argR, the gene for the arginine repressor. Here we have compared the binding of the two repressors to the operator sites of argF (ARG boxes). In DNase I footprinting and gel retardation experiments with argF ARG boxes we have shown that the arginine repressor of E. coli K12 bound to arginine (ArgRK-arg) has a greater affinity than the arginine repressor of E. coli B bound to arginine (ArgRB-arg), whereas free ArgRB (ArgRBf) has a much stronger affinity than free ArgRK (ArgRKf). The stronger binding of ArgRBf can explain the repression seen in E. coli B during arginine-limited growth and indicates that ArgRBf, but not ArgRKf, is able to repress enzyme synthesis under physiological conditions. The weaker repression of E. coli B than of E. coli K12 seen in the presence of arginine can be explained by the lower affinity of ArgRB-arg for operator sites as compared to ArgRK-arg. Another contributing cause for the weaker repression is the reduction of ArgRBf concentration due to autoregulation of the gene for the repressor. Thus the combined effects of repression by ArgRBf, but not ArgRKf, with the weaker repression by ArgRB-arg as compared to ArgRK-arg, convert the arginine dependent regulation in E. coli K12 to arginine independent regulation in E. coli B.

Codon reading scheme in Mycoplasma pneumoniae revealed by the analysis of the complete set of tRNA genes.

The 33 genes encoding the complete set of tRNA species in Mycoplasma pneumoniae have been cloned and sequenced. They are organized into 5 clusters in addition to 9 single genes. No redundant gene was found, indicating that 33 tRNAs correspond to 32 different anticodons and decode all 62 codons used in this organism. There is only one single tRNA for each of the Ala, Leu, Pro, and Val family boxes. Therefore, a simplified decoding system resembling that recently described for Mycoplasma capricolum (1) has to also exist in M.pneumoniae. However, analysis of the anticodon set and codon usage revealed features characteristic of the latter: (i) there is no obvious preference toward AT rich synonymous codons, (ii) CGG codons are assigned for arginine and are translated by tRNA Arg(UCG), and (iii) CNN or GNN anticodons are encountered in the Ser, Thr, Arg, and Gly family boxes. We thus propose that this codon-anticodon recognition pattern has emerged in the 'M.pneumoniae cluster' under a genomic economization strategy but without the influence of AT pressure.

Binding of the arginine repressor of Escherichia coli K12 to its operator sites

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of l-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70° and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressers studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.

Arginine regulon of Escherichia coli K-12 : A study of repressor-operator interactions and of in vitro binding affinities versus in vivo repression

The 12 genes which in E. coli K-12 constitute the arginine regulon are organized in nine transcriptional units all of which contain in their 5′ non-coding region two 18 bp partially conserved imperfect palindromes (ARG boxes) which are the target sites for binding of the represser, a hexameric protein. In vitro binding experiments with purified repressor (a gift from W. K. Maas) were performed on the operator sites of four genes, argA, argD, argF, argG, and of two operons, carAB and the bipolar argECBH cluster. A compilation of results obtained by DNase I and hydroxyl radical footprinting clearly indicates that in each case the repressor binds symmetrically to four helical turns covering adjacent pairs of boxes separated by 3 bp, but to one face of the DNA only. Methylation protection experiments bring to light major base contacts with four highly conserved G residues symmetrically distributed in four consecutive major grooves. Symmetrical contacts in the minor groove with A residues have also been identified. Stoichiometry experiments suggest that a single hexameric repressor molecule binds to a pair of adjacent ARG boxes. Although the wildtype operator consists of a pair of adjacent ARG boxes separated by 3 bp (except argR where there are only 2 bp), repressor can bind to a single box but with a greatly reduced affinity. Therefore, adjacent boxes behave co-operatively with respect to the Arg repressor binding, in the sense that the presence of one box largely stimulates the binding of the properly located second box. The optimal distance separating two boxes is 3 bp, but one bp more or less does not abolish this stimulation effect. However, it is completely abolished by the introduction of two or more additional bp unless a full helical turn is introduced. Large variations in the in vivo repression response between individual arginine genes or a wild-type gene and cognate O c type mutants are not reflected by similar differences in the in vitro binding results where only small differences are observed. The significance of this lack of correlation is discussed.

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 M r subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/ carAB operator is 6 × 10 −12 m. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive “downstream” carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined “ARG box” sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A · T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A · T-rich regions rather than on the specific nucleotide sequence.

Sequences required for regulation of arginine biosynthesis promoters are conserved between Bacillus subtilis and Escherichia coli

The region required for regulation of a previously characterized arginine-regulatable promoter upstream from the argC gene in the argCAEBD-cpa-argF cluster of Bacillus subtilis was defined by integration of argC-lacZ translational fusions into the chromosome at a site distant from the arginine loci. Some sequence similarity was detected between the argC regulatory region and the well-characterized Escherichia coli arginine operators (ARG boxes). This similarity was shown to be functional in vivo in that the B. subtilis repressor regulated the E. coli arginine genes, but the E. coli repressor, even when encoded by a multicopy plasmid, could not repress the B. subtilis argC promoter. In vitro binding studies using purified repressors on DNA fragments encoding operators from both E. coli and B. subtilis demonstrated interactions by both proteins.

Molecular interactions in the control region of the carAB operon encoding Escherichia coli carbamoylphosphate synthetase

The control region of the carAB operon, encoding carbamoylphosphate synthetase, comprises two tandem promoters (P1, upstream and P2, downstream) located 67 base-pairs apart and repressed respectively by pyrimidines and arginine. RNA polymerase and pure arginine repressor bind to the P2 region in mutually exclusive ways. Repressor protects the two adjacent palindromic ARG boxes overlapping P2 against DNase I. Binding of RNA polymerase to P1 is abnormal; the region protected against DNase I is shifted upstream by about 20 nucleotides with respect to the position expected from the transcription startpoint. This pattern is not due to interference with polymerase binding at P2, since it is observed also in the presence of repressor and on an isolated P1 region. Binding of RNA polymerase is relatively weak and heparin-sensitive suggesting that, in vivo, an ancillary factor is required to promote the formation of an open complex. S 1 nuclease mapping experiments show that the simultaneous presence of pyrimidines and arginine represses the downstream arginine-specific promoter (P2) more efficiently than arginine alone. This effect is not due to a direct regulatory interaction between pyrimidines and P2, since it is not observed when P1 is inactivated by insertion mutations or partial deletion. It has been shown that transcription initiated at P1 can proceed even when arginine represses P2. We therefore suggest that P2 operator-arginine repressor complex is destabilized by RNA polymerase binding at P1 or transcription from P1. We describe a novel technique to select for expression-down mutants in a lac fusion context.

CarP, a novel gene regulating the transcription of the carbamoylphosphate synthetase operon of Escherichia coli

The carAB operon, encoding carbamoylphosphate synthetase (CPSase; EC 6.3.5.5) is transcribed from two tandem promoters. The upstream promoter (P1) is controlled by pyrimidines and the downstream promoter (P2) is controlled by arginine. We have isolated a new type of constitutive mutation ( carP) that specifically affects the control of the pyrimidine-sensitive promoter but does not appear to influence other genes of the pyrimidine pathway. The carP mutation acts in trans and is dominant, which suggests that the carP product is an activator of car transcription. The downstream promoter P2, which is repressed by arginine, overlaps two operator modules characteristic of the arginine regulon. We have isolated two operator-constitutive mutations that specifically affect P2; both map in the upstream ARG box at a strongly conserved position.

Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene.

In Pseudomonas aeruginosa PAO the anabolic ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) is the product of the argF gene and the only arginine biosynthetic enzyme whose synthesis is repressible by arginine. We have determined the complete nucleotide sequence of the argF gene including its promoter-control region. The deduced amino acid sequence of the anabolic OTCase consists of 305 residues (Mr 33,924), and this was confirmed by the N-terminal amino acid sequence, the total amino acid composition, and the subunit Mr of the purified enzyme. The native anabolic OTCase (Mr 110,000 to 125,000) was found to be a trimer by cross-linking experiments. P. aeruginosa also has a catabolic OTCase (the arcB gene product), which catalyzes the reverse reaction of the anabolic conversion. At the nucleotide sequence level, the P. aeruginosa argF gene had 52.4% identity with the arcB gene. The Escherichia coli argF and argI genes, which code for anabolic OTCase isoenzymes, had 47.3 and 44.9% identity, respectively, with the P. aeruginosa argF sequence. This suggests that these four genes have evolved from a common ancestral gene. The arcB gene appears to be more closely related to the E. coli argF gene than to the P. aeruginosa argF gene. Two transcripts (mRNA-1, mRNA-2) of the P. aeruginosa argF gene were identified by S1 mapping. The transcription initiation site for mRNA-1 was preceded by sequences having partial homology with the E. coli -35 and -10 consensus promoter sequences. No sequence similar to consensus promoters of enteric bacteria was found upstream of the 5' end of mRNA-2. E. coli carrying a P. aeruginosa argF+ recombinant plasmid produced mRNA-1 with low efficiency but no (or very little) mRNA-2. Arginine repressed argF transcription in P. aeruginosa. In the argF promoter region no sequence homologous to the "arg box" (arginine operator module) of E. coli was found. The mechanism of arginine repression in P. aeruginosa thus appears to be different from that in E. coli.

Nucleotide sequence of the argR gene of Escherichia coli K-12 and isolation of its product, the arginine repressor.

In Escherichia coli, the arginine repressor, the product of the argR gene, in conjunction with L-arginine controls the synthesis of the enzymes of arginine biosynthesis. We describe the nucleotide sequence of the argR gene, including its control region, and show that formation of the repressor is autoregulated. The argR control region contains two promoters, one of which overlaps the operator site and, as with other arg genes, consists of two adjacent palindromic sequences ("ARG boxes"). The arginine repressor protein and an arginine repressor-beta-galactosidase fusion protein were purified, and the amino acid sequence of the N-terminal end of the repressor protein portion of the fusion protein was determined. Antibodies prepared against the fusion protein react with the repressor. The repressor is precipitable by L-arginine, which facilitates its purification. The native repressor is a hexamer with a molecular weight of 98,000; its monomeric subunit has a molecular weight of 16,500. To verify its properties postulated from genetic studies, we show that in the presence of L-arginine, repressor inhibits transcription of argF and binds to the ARG boxes of argF and argR.