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The mechanism by which the cyclic AMP receptor protein, CRP, stimulates transcription of the Escherichia coli araBAD promoter was studied in vitro. Under one set of conditions, CRP stimulated by eightfold the rate of RNA polymerase open complex formation on supercoiled DNA template containing the normal wild-type araBAD regulatory region. Since previous studies in vivo had identified an upstream site termed araO2 that is involved in both repression and in the CRP requirement for PBAD induction, we performed similar experiments in vitro. Deletion of araO2 or alterations of its orientation with respect to the araI site by half integral numbers of turns greatly reduced the CRP requirement for induction of PBAD. Linearizing the DNA has the same effect as deleting araO2 from the supercoiled DNA template. The similarity of conditions that relieve the classical repression of PBAD in vivo and the conditions that eliminate the requirement for CRP for maximal activity in vitro suggest a close relationship between repression in the ara system and the role of CRP. At lower concentrations of AraC protein and slightly different conditions than those used in the above-mentioned experiments, CRP does stimulate transcription from linear or supercoiled templates lacking araO2. On linear DNA under these conditions, one dimer of AraC protein binds to linear araPBAD DNA, but is incapable of stimulating transcription without the additional binding of CRP. The responses of the ara system under the second set of conditions are unlike its behavior in vivo.
J Mol Biol 1986 Apr 05
PMID:Transcription of Escherichia coli ara in vitro. The cyclic AMP receptor protein requirement for PBAD induction that depends on the presence and orientation of the araO2 site. 301 84

The rhaC gene, whose product is the positive activator of the genes required for L-rhamnose utilization, has been cloned along with the rhamnose structural genes. The rhaC sequence shows two partially overlapping reading frames, encoding two proteins of molecular weight 32,000 and 35,000 RhaS and RhaR. Both proteins show significant homology to AraC, the positive activator of the arabinose operon. S1 mapping located transcriptional start points and showed that RhaR, and possibly RhaS, positively regulate transcription from the structural gene promoters as well as transcription from their own promoter. In-vivo dimethyl sulfate footprinting and DNase I footprinting indicate that the RhaR protein may bind to DNA elements upstream from its RNA polymerase binding site.
J Mol Biol 1987 Aug 20
PMID:Positive regulation of the Escherichia coli L-rhamnose operon is mediated by the products of tandemly repeated regulatory genes. 331 63

Experiments on the AraC regulatory protein of Escherichia coli suggest a mechanism that DNA-binding proteins can use to reduce potentially drastic alterations in their affinity for DNA resulting from changes in salt concentration. Measurement of the net number of ions apparently displaced as AraC protein binds DNA and of fluorescence changes in the protein lead to the following picture. About 14 ions are displaced from the DNA as the protein binds the araI site. As the protein binds the DNA, however, it undergoes a conformational change and binds about ten ions. Consequently, the net order of the reaction is reduced from 15th to about fourth order in salt concentration.
J Mol Biol 1987 Jun 05
PMID:Equilibrium DNA-binding of AraC protein. Compensation for displaced ions. 365 30

Repression of the Escherichia coli araBAD promoter, PBAD, was studied using a mutant PBAD promoter (cip-5) that is expressed in the absence of the two proteins required for PBAD induction, AraC protein and the cyclic AMP receptor protein (CRP-cAMP). Like the wild type promoter, cip-5 was repressed by AraC protein, and this repression required a site well upstream of the transcriptional start site. cip-5 was used to determine whether repression results from interference with the functioning of either AraC protein at araI and/or CRP-cAMP. Repression of cip-5 was eliminated by a point mutation within the AraC protein binding site araI but was not affected in the absence of CRP-cAMP. These results suggest that repression involves an interaction between two AraC protein binding sites located over 200 nucleotides apart. Our results also suggest that the majority of the CRP requirement for PBAD is a result of PBAD repression. When repression was abolished by deletion of the araO2 site, the requirement for CRP-cAMP in PBAD induction was greatly reduced.
J Mol Biol 1984 Nov 25
PMID:Upstream repression and CRP stimulation of the Escherichia coli L-arabinose operon. 639 69

We investigated the mechanism of action of 2-aminopurine (Apur) in eucaryotic cells. By analogy with studies in procaryotic systems, the base analog is presumed to incorporate into DNA predominantly opposite T where, upon subsequent DNA replication, it can mispair with C, inducing an A:T leads to G:C transition. This model predicts that Apur-induced mutagenesis will be enhanced by factors that favor formation of Apur-C mispairs, e.g., high levels of dCTP or low levels of TTP. We describe the use of a mutant T-lymphosarcoma cell line, AraC-6-1, which has an abnormally high dCTP pool and a low TTP pool, to test this prediction. AraC-6-1 cells were three- to fivefold more mutable by Apur than their parental cell line, NSU-1. This enhanced mutability by Apur could not be explained by altered incorporation of 3H-labeled Apur, by generally impaired ability to repair DNA damage, or by a direct effect of Apur on the endogenous deoxynucleotide pools. The addition of 10 microM thymidine to the growth medium of AraC-6-1 cells lowered their high dCTP pool (two- to threefold), raised the TTP pool (two- to threefold), and abolished their enhanced mutability by Apur. Further manipulation to produce an abnormally high TTP/dCTP ratio suppressed Apur-induced mutagenesis (8- to 10-fold) in both AraC-6-1 and NSU-1 cells. These observations support the hypothesis that Apur induces A:T leads to G:C transitions in mammalian cells by a mispairing mechanism.
Mol Cell Biol 1982 Sep
PMID:Mechanism of 2-aminopurine mutagenesis in mouse T-lymphosarcoma cells. 698 47

Urease activity is produced by members of the family Enterobacteriaceae that contain the plasmid-encoded urease locus only when urea is present in the growth medium. The plasmid-encoded urease locus contains seven tandem urease structural and accessory genes (ureDABCEFG). Previously we showed that transcription of the first gene in this cluster, ureD, is initiated at a urea-dependent promoter (ureDp). Expression from ureDp requires the product of ureR, which is transcribed divergently from the plasmid-encoded ureDABCEFG. From DNA sequence analysis, UreR is predicted to be a 34 kDa protein with identity to the AraC family of transcriptional activators. In this report we demonstrate that there are two additional urea and UreR-dependent promoters within the plasmid-encoded urease locus: ureRp and ureGp. A low-level constitutive promoter was also identified upstream of ureE (ureEp). Three major mRNA transcripts were induced when urea was present in the growth medium: a transcript containing ureDABCEF, a transcript corresponding to ureG, and a transcript corresponding to ureR. These results indicate that expression of each of the plasmid-encoded urease genes is transcriptionally regulated in response to urea and suggest that there is autogenous regulation of ureR. Therefore UreR is one of three AraC family members described thus far that are positively auto-regulated.
Mol Microbiol 1995 Apr
PMID:UreR activates transcription at multiple promoters within the plasmid-encoded urease locus of the Enterobacteriaceae. 765 Nov 32

A genetic method was developed to determine, in proteins, areas which are tolerant of insertions and deletions. Attractive candidates for these areas are linker regions. Such a region was found to include positions 171 to 178 in the Escherichia coli regulatory protein AraC. Independent biochemical methods identified amino acid residues 11 to 170 as the minimal dimerization domain of AraC, and amino acid residues 178 to 286 out of the 291 residue protein as the minimal DNA-binding domain. Hence, by both the genetic and biochemical approaches, the interdomain linking region was determined to include amino acid residues 171 to 177. The properties of altered proteins were examined using templates with AraC half-sites more widely separated than in the wild-type case. Both AraC protein containing an insertion in the interdomain linker region and a protein consisting of the minimal functional dimerization and DNA-binding domains separated by a 39 amino acid residue linker were able to bind to and function on such a DNA site. In vitro, the proteins with longer linkers bound substantially more stably than wild-type AraC to the DNA containing half-sites for AraC separated by an extra two helical turns of DNA. In vivo on an ara promoter with the more widely separated AraC half-sites, the proteins could activate transcription much better than wild-type AraC.
J Mol Biol 1994 Sep 30
PMID:Reaching out. Locating and lengthening the interdomain linker in AraC protein. 793 93

The nickel metalloenzyme urease catalyses the hydrolysis of urea to ammonia and carbamate, and thus generates the preferred nitrogen source of many organisms. When produced by bacterial pathogens in either the urinary tract or the gastroduodenal region, urease acts as a virulence factor. At both sites of infection urease is known to enhance the survival of the infecting bacteria. Ammonia resulting from the action of urease is believed to increase the pH of the environment to one more favourable for growth, and to injure the surrounding epithelial cells. In addition, in the urinary tract urease activity can result in the formation of urinary calculi. Bacterial urease gene clusters contain from seven to nine genes depending upon the species. These genes encode the urease structural subunits and accessory polypeptides involved in the biosynthesis of the nickel metallocentre. So far, three distinct mechanisms of urease gene expression have been described for ureolytic bacteria. Some species constitutively produce urease; some species produce urease only if urea is present in the growth medium; and some species produce urease only during nitrogen-limiting growth conditions. For either the urea-inducible genes or the nitrogen-regulated genes transcription appears to be positively regulated. In the nitrogen-regulated systems, urease gene expression requires Nac (nitrogen assimilation control), a member of the LysR family of transcriptional activators. Urea dependent expression of urease requires UreR (urease regulator), a member of the AraC family of transcriptional activators. An evolutionary tree for urease genes of eight bacterial species is proposed.
Mol Microbiol 1993 Sep
PMID:Bacterial ureases: structure, regulation of expression and role in pathogenesis. 793 18

TOL plasmid pWW0 of Pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. The upper pathway operon encodes the enzymes for the oxidation of toluene/xylenes to benzoate/toluates, and the metacleavage pathway operon encodes the enzymes for the further oxidation of these compounds to Krebs cycle intermediates. Their expression is controlled by the gene products of two divergently transcribed regulatory genes, xyIR and xyIS. The XyIR protein, which belongs to the NtrC family of regulators, is expressed from two tandem promoters and autoregulates its synthesis. XyIR stimulates transcription from the xyIS gene promoter (Ps) and the upper pathway operon promoter (Pu) in the presence of pathway substrates. Both promoters are sigma 54 dependent, and Pu also requires the presence of integration host factor (IHF) for activation of transcription. Binding sites for XyIR and IHF in the Pu promoter and for XyIR in the Ps promoters have been defined. The XyIS protein, which belongs to the AraC family of regulators, stimulates transcription from the meta-cleavage pathway operon promoter (Pm) in the presence of benzoates. The effector binding pocket and DNA-binding region of XyIS have been defined through the isolation of mutants that exhibit altered effector specificity and modified transcriptional patterns, respectively. Expression of the meta-cleavage pathway operon is also induced by xylene-activated XyIR protein via a cascade regulatory system in which this protein, in combination with sigma 54, stimulates the expression from the xyIS promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Microbiol 1993 Sep
PMID:Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways. 793 20

Previous work has indicated that the RhaS protein directly activates the L-rhamnose catabolic operon, rhaBAD, and that the likely RhaS binding site lies downstream of position -84 relative to the rhaBAD transcription start point. Biochemical analysis of RhaS binding to this DNA site had not been possible due to the extreme insolubility of overproduced RhaS protein. Here we have been able to analyze directly the DNA binding properties of RhaS by developing a method to refold insoluble RhaS protein into a form with specific DNA binding activity. We found that active RhaS protein could be recovered only if the renaturation reaction was performed in the presence of DNA. We also found that the recovery of DNA-binding activity from the related AraC protein, after denaturation in urea, was dependent upon added DNA. To test the specificity of the recovered RhaS DNA-binding activity, and to define the binding site for comparison with other AraC family binding sites, we then investigated the details of the RhaS binding site. Using refolded RhaS protein in a DNase footprinting assay, we found that RhaS protects a region of the rhaBAD promoter from position -83 to -28. Analysis of the effects of single base mutations in the rhaBAD promoter region indicates that RhaS binds to an inverted repeat of two 17 bp half-sites separated by 16 bp, located between -81 and -32 relative to the rhaBAD transcription start site.
J Mol Biol 1994 Nov 11
PMID:DNA-dependent renaturation of an insoluble DNA binding protein. Identification of the RhaS binding site at rhaBAD. 796 3


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