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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

PobR is a transcriptional activator required for the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase. The pobA and pobR genes are divergently transcribed and separated by 134 bp in the Acinetobacter calcoaceticus chromosome. Primer extension analysis revealed that the pobA transcript begins 22 bp upstream from the structural gene and the pobR transcript begins 69 bp upstream from the regulatory gene. This arrangement requires superimposition of the -10 base pair and -35 base pair RNA polymerase-binding sites for the respective genes. Expression of a pobR-lacZ fusion was found to be repressed three- to fourfold by pobR when the functional gene was carried in trans on a plasmid. The pobR gene was placed under control of a lac promoter in an expression vector, and the recombinant plasmid inducibly expressed high levels of PobR in Escherichia coli. Cell extracts containing this protein were used to conduct gel mobility shift analyses. PobR binds specifically to DNA in the pobA-pobR intergenic region, and this binding does not appear to be influenced by p-hydroxybenzoate, the inducer of pobA expression. DNase I footprinting indicates that the DNA-binding site for PobR extends from about 10 bp to about 45 bp downstream from the site of the beginning of the pobR transcript. Within this putative operator is a region of inverted symmetry. Evidently, interaction of the inducer with the PobR-operator complex triggers elevated expression of pobA, beginning at a position separated by 55 bp of DNA. The general mechanisms by which PobR exerts transcriptional control resemble those that typify the LysR family of transcriptional activators, a group from which PobR is evolutionarily remote.
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PMID:Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus. 802 Dec 13

We have identified pobR, a gene encoding a transcriptional activator that regulates expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase (PobA) in Acinetobacter calcoaceticus ADP1. Inducible expression of cloned pobA in Escherichia coli depended upon the presence of a functional pobR gene, and mutations within pobR prevented pobA expression in A. calcoaceticus. A pobA-lacZ operon fusion was used to demonstrate that pobA expression in A. calcoaceticus is enhanced up to 400-fold by the inducer p-hydroxybenzoate. Inducer concentrations as low as 10(-7) M were sufficient to elicit partial induction. Some structurally related analogs of p-hydroxybenzoate, unable to cause induction by themselves, were effective anti-inducers. The nucleotide sequence of pobR was determined, and the activator gene was shown to be transcribed divergently from pobA; the genes are separated by 134 DNA base pairs. The deduced amino acid sequence yielded a polypeptide of M(r) = 30,764. Analysis of this sequence revealed at the NH2 terminus a stretch of residues with high potential for forming a helix-turn-helix structure that could serve as a DNA-binding domain. A conservative amino acid substitution (Arg-61-->His-61) in this region inactivated PobR. The primary structure of PobR appears to be evolutionarily distinct from the four major families of NH2-terminal helix-turn-helix containing bacterial regulatory proteins that have been identified thus far.
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PMID:Identification of the transcriptional activator pobR and characterization of its role in the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase in Acinetobacter calcoaceticus. 833 Oct 77

Analysis of spontaneous mutations in Acinetobacter calcoaceticus revealed a 1237 bp insertion sequence named IS1236 and possessing a nucleotide sequence resembling those of members of the lS3 family. The chromosome of A. calcoaceticus strain ADP1 contains seven copies of IS1236 which appears to insert preferentially into pobR, the transcriptional activator of the structural gene for p-hydroxybenzoate hydroxylase. IS1236 creates tandem 3 bp DNA duplications flanking the sites of its insertion in pobR. Different duplication patterns are found following insertion of IS1236 into pcaH, a structural gene for protocatechuate 3,4-dioxygenase. Therefore the insertion of properties of IS1236 appear to be influenced by its DNA target. Amino acid sequences associated with the apparent transposase function have been conserved in ORFB of IS1236 whereas the presumed DNA-binding helix-turn-helix region of IS1236 ORFA exhibits substantial amino acid sequence divergence from its IS3 counterparts. IS1236 ORFA and ORFB coding sequences overlap considerably, and sequence evidence indicates mechanisms for ORFB expression in IS1236 may resemble those employed by other members of the IS3 family. Portions of the IS1236 terminal repeats exhibit substantial sequence divergence from other members of the IS3 family, but evolution appears to have conserved a mechanism preventing expression of the insertion sequence genes as a consequence of transcriptional readthrough.
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PMID:IS1236, a newly discovered member of the IS3 family, exhibits varied patterns of insertion into the Acinetobacter calcoaceticus chromosome. 875 45

We present a procedure for efficient random mutagenesis of selected genes in a bacterial chromosome. The method combines PCR replication errors with the uptake of PCR-amplified DNA via natural transformation. Cloning of PCR fragments is not required, since mutations are transferred directly to the chromosome via homologous recombination. Random mutations were introduced into the Acinetobacter chromosomal pobR gene encoding the transcriptional activator of pobA, the structural gene for 4-hydroxybenzoate 3-hydroxylase. Mutant strains with strongly reduced PobR activity were selected by demanding the inability to convert 4-hydroxybenzoate to a toxic metabolite. Of spontaneous pobR mutants, 80% carry the insertion element IS1236, rendering them inappropriate for structure-function studies. Transformation with Taq-amplified pobR DNA increased the mutation frequency 240-fold and reduced the proportion of IS1236 inserts to undetectable levels. The relative fidelity of Pfu polymerase compared with Taq polymerase was illustrated by a reduced effect on the mutation frequency; a procedure for rapid assessment of relative polymerase fidelity in PCR follows from this observation. Over 150 independent mutations were localized by transformation with DNA fragments containing nested deletions of wild-type pobR. Sequence analysis of 89 of the mutant pobR alleles showed that the mutations were predominantly single-nucleotide substitutions broadly distributed within pobR. Promoter mutations were recovered, as were two mutations that are likely to block pobR translation. One-third of the recovered mutations conferred a leaky or temperature-sensitive phenotype, whereas the remaining null mutations completely blocked growth with 4-hydroxybenzoate. Strains containing two different nonsense mutations in pobR were transformed with PCR-amplified DNA to identify permissible codon substitutions. Independently, second-site suppressor mutations were recovered within pcaG, another member of the supraoperonic pca-qui-pob cluster on the Acinetobacter chromosome. This shows that combining PCR mutagenesis with natural transformation is of general utility.
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PMID:Combining localized PCR mutagenesis and natural transformation in direct genetic analysis of a transcriptional regulator gene, pobR. 920 43

We report the cloning and analysis of a gene and its cognate regulatory element from a member of the Azotobacteriaceae which are involved in the breakdown of an aromatic compound. The genes from Azotobacter chroococcum encoding p-hydroxybenzoate hydroxylase (pobA) and its regulatory protein (pobR) were cloned from a genomic library and sequenced. Sequence analysis of pobA revealed homology with other bacterial p-hydroxybenzoate hydroxylase enzymes. Residues essential to the structure and function of the enzyme have been conserved. The pobR gene encodes a DNA binding regulatory protein with similarity to proteins from the AraC/XylS family of transcriptional activators. A fragment containing both pobA and pobR was cloned into pUC19 and p-hydroxybenzoate hydroxylase activity was induced in Escherichia coli by the addition of p-hydroxybenzoate. A frame-shift mutation introduced into the pobR gene prevented expression of p-hydroxybenzoate hydroxylase, indicating that PobR is the protein required for transcription of pobA. Interestingly, A. chroococcum PobR has no homology to the PobR protein that is the transcriptional activator of pobA in Acinetobacter strain ADP1, a protein that is homologous to the IclR family of transcriptional regulators. However, PobR from A. chroococcum is homologous to several other proteins, suggesting that these proteins will also function as transcriptional activators of pobA.
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PMID:Analysis of the pobA and pobR genes controlling expression of p-hydroxybenzoate hydroxylase in Azotobacter chroococcum. 1124 81

As other environmental bacteria, Cupriavidus necator JMP134 uses benzoate as preferred substrate in mixtures with 4-hydroxybenzoate, strongly inhibiting its degradation. The mechanism underlying this hierarchical use was studied. A C. necator benA mutant, defective in the first step of benzoate degradation, is unable to metabolize 4-hydroxybenzoate when benzoate is also included in the medium, indicating that this substrate and not one of its catabolic intermediates is directly triggering repression. Reverse transcription polymerase chain reaction analysis revealed that 4-hydroxybenzoate 3-hydroxylase-encoding pobA transcripts are nearly absent in presence of benzoate and a fusion of pobA promoter to lacZ reporter confirmed that benzoate drastically decreases the transcription of this gene. Expression of pobA driven by a heterologous promoter in C. necator benA mutant, allows growth on 4-hydroxybenzoate in presence of benzoate, overcoming its repressive effect. In contrast with other bacteria, regulators of benzoate catabolism do not participate in repression of 4-hydroxybenzoate degradation. Moreover, the effect of benzoate on pobA promoter can be observed in heterologous strains with the sole presence of PobR, the transcriptional activator of pobA gene, indicating that PobR is enough to fully reproduce the phenomenon. This novel mechanism for benzoate repression is probably mediated by direct action of benzoate over PobR.
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PMID:Strict and direct transcriptional repression of the pobA gene by benzoate avoids 4-hydroxybenzoate degradation in the pollutant degrader bacterium Cupriavidus necator JMP134. 2145 7