Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used a sequence-specific DNA binding protein to examine transcription elongation and termination by mammalian RNA polymerase III (polIII). The Escherichia coli lac repressor protein, bound to its cognate operator site positioned between the 3' end of the coding region and the termination site of a human tRNA gene, conditionally blocked transcription elongation by polIII in vitro in HeLa cell nuclear extracts. Arrest of elongation by polIII dramatically reduced overall levels of transcription and directed the synthesis of shortened transcripts, consistent with a block to polIII elongation at the boundary of the repressor/DNA complex. Removal of template-bound repressor with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG) allowed extension of nascent transcripts and restored transcriptional activity. Moreover, a subset of transcription complexes were shown to be capable of transcribing through the repressor obstacle. lac repressor positioned just downstream of the natural termination site effected the premature termination of transcription but otherwise had no affect on the overall level of transcription. Our findings demonstrate that elongation and termination by mammalian polIII can be modulated in vitro by a heterologous sequence-specific DNA binding protein. Moreover, the ability to selectivity arrest elongation by polIII at defined positions within the tRNA gene transcription unit has permitted the identification of discrete functional properties of paused mammalian polIII ternary complexes.
J Mol Biol 1994 Dec 16
PMID:RNA chain elongation and termination by mammalian RNA polymerase III. Analysis of tRNA gene transcription by imposing a reversible factor-mediated block to elongation using a sequence-specific DNA binding protein. 799 Jan 36

The cI repressor protein (cI) maintains bacteriophage lambda in the lysogenic state in infected Escherichia coli cells by binding cooperatively to three tandemly repeated sequences comprising the right operator (OR). Cooperative interactions occur between alternate pairs of cI dimers bound to adjacent sites. Although crystallographic studies have revealed the structure of the DNA in the 92 amino acid residue amino-terminal fragment-OL1 complex, the structure of the DNA within the OR-cI complex with intact, cooperatively bound cI has not been described. In this study, the structure of the DNA within OR was quantitatively examined using sequence and structure-dependent nuclease cleavage patterns as a function of cI binding. The cooperative binding of cI to OR1 and OR2 induces a conformational change in the DNA of OR3 that is detectable by both DNase I and 5-phenyl-1,10-phenanthroline. Hydroxyl radical footprinting indicates the presence of an "A-tract" between OR1 and OR2 at the site of a run of four adenine-thymine base-pairs, implying a stable bend between the sites of approximately 18 degrees. 5-Phenyl-1,10-phenanthroline footprinting reports conformational changes within the central base-pairs of all three sites that is dependent upon the sequence-specific binding of cI. The observed conformational changes are more extensive within OR2 and OR3 compared with OR1, consistent with an "induced-fit" model of sequence-specific recognition. A number of changes in nuclease reactivity within the individual binding sites were quantitatively correlated with cI binding at the other sites within OR. These results demonstrate that changes in the DNA structure are propagated among the sites in response to the binding of cI and imply a role for DNA sequence-dependent conformational changes in the mechanisms of both the intrinsic and cooperative binding reactions of cI to OR.
J Mol Biol 1994 Dec 16
PMID:DNA conformational changes associated with the cooperative binding of cI-repressor of bacteriophage lambda to OR. 799 Jan 37

Arginine biosynthesis in Escherichia coli is negatively regulated by a hexameric repressor protein, encoded by the gene argR and the corepressor arginine. By hydroxylamine mutagenesis two types of argR mutants were isolated and mapped. The first type is transdominant. In heterodiploids, these mutant polypeptides reduce the activity of the wild-type repressor, presumably by forming heteropolymers. Four mutant repressor proteins were purified. Two of these map in the N-terminal half of the protein. Gel retardation experiments showed that they bind poorly to DNA, but they could be precipitated by L-arginine at the same concentration as the wild-type repressor. The other two mutant repressors map in the C-terminal half of the protein. They are poorly precipitated by L-arginine and they bind poorly to DNA. In addition, one of these mutants appears to exist as a dimer. The second type of argR mutant repressor consists of super-repressors. Such mutants behave as arginine auxotrophs as a result of hyper-repression of arginine biosynthetic enzymes. They map at many locations throughout the argR gene. Three arginine super-repressor proteins were purified. In comparison with the wild-type repressor, two of them were shown to have a higher DNA-binding affinity in the absence of bound arginine, while the third was shown to have a higher DNA-binding affinity when bound to arginine.
Mol Microbiol 1994 Aug
PMID:Mutational analysis of the arginine repressor of Escherichia coli. 799 72

The complete nucleotide sequence of the 8.3 kilobase operon of the enterobacterial virulence plasmid pColV-K30, which encodes a high-affinity iron transport system mediated by the hydroxamate siderophore aerobactin, has been determined. The region includes five open reading frames which correspond to the genes iucA, iucB, iucC and iucD, encoding the enzymes of the biosynthetic pathway for aerobactin, and iutA for the outer membrane receptor of ferri-aerobactin complexes. The sequences of the iucABCD genes are tightly coupled without any intervening non-coding sequence. The predicted secondary mRNA structures at the gene junctions within the iucABCD cluster, along with their codon usage, may account for the differential expression of each of the protein products, as observed in vivo with minicells. The genes iucA and iucC, which determine the two subunits of the aerobactin synthetase complex, showed a considerable homology within three stretches of their amino acid sequence. A potential operator sequence (iron box) for the binding of the iron(II)-responsive Fur repressor protein was found within the iucA coding region, suggesting that the operon is subjected to an additional level of transcriptional repression by iron (II).
J Mol Biol 1994 Apr 29
PMID:The organization of intercistronic regions of the aerobactin operon of pColV-K30 may account for the differential expression of the iucABCD iutA genes. 800 7

Although iron is an essential nutrient, its toxicity at high levels necessitates regulated transport. In Gram-negative bacteria a central target for regulation is the TonB protein, an energy transducer that couples the cytoplasmic membrane proton motive force to active transport of (FeIII)-siderophore complexes across the outer membrane. We have previously demonstrated the threefold repression of tonB transcription by excess iron in the presence of Fur repressor protein under aerobic conditions. In this report, we examine tonB regulation under anaerobic conditions where the solubility of iron is not a limiting factor and, presumably, siderophore-mediated transport is not required. Under these conditions, tonB transcription is repressed at least 10-fold by excess iron in the presence of Fur, but can be fully derepressed in the absence of Fur. Based on several lines of evidence, this anaerobic repression is not due to increased negative supercoiling as previously postulated. Our results rule out both supercoiling-mediated decreased promoter function and increased Fur binding as mediators of anaerobic repression. Under iron-limiting anaerobic conditions tonB expression is as high or higher than under iron-limiting aerobic conditions, suggesting that promoter function has not decreased anaerobically. Furthermore, under anaerobic conditions in tonB+ strains, tonB promoter function is insensitive to the gyrase inhibitor novobiocin and to changes in medium osmolarity and temperature, three conditions known to change levels of supercoiling. We also rule out effects of mutations in arcA or fnr as mediators of anaerobic repression. Results from in vivo dimethyl sulphate protection foot-printing indicate that Fur binds to an operator site between the -10 and -35 regions of the promoter, but not to a less homologous operator site centered at +26. The binding is, if anything, weaker under anaerobic conditions, indicating that anaerobic repression is not mediated through Fur. Additional changes in the in vivo footprint upstream from the promoter implicate a second factor in tonB anaerobic repression. Together, these results suggest that the mechanism responsible for this regulation (and, by analogy, that of other anaerobically repressed, iron-regulated genes such as cir, exbB, and fhuA) is a novel one.
Mol Microbiol 1994 Mar
PMID:Repression of tonB transcription during anaerobic growth requires Fur binding at the promoter and a second factor binding upstream. 802 70

Hydration forces are believed to play a determining role in protein folding. We have examined the contribution of water for the stability of the native dimer state of Arc repressor, a DNA-binding protein. Hydrostatic pressure was utilized to convert Arc repressor protein from a native state to a denatured, molten-globule state at decreasing concentrations of water. The volume change associated with Arc denaturation fell linearly with the increase in concentration of glycerol, whereas the free energy of the reaction increased. The pressure that promotes 50% denaturation (p1/2) increased in direct proportion to the concentration of glycerol or the decrease of water. Extrapolated to zero concentration of water, the data indicate that pressure denaturation would not occur without water. It is concluded that water plays a crucial role in decreasing the stability of a protein to a level that is compatible with its biological properties.
J Mol Biol 1994 Jul 15
PMID:Arc repressor will not denature under pressure in the absence of water. 802 2

Amber mutations have been constructed at 328 positions, corresponding to residues 2 to 329 in the E. coli lac repressor protein. Synthetic and naturally occurring nonsense suppressors have been used to insert, in series, 12-13 amino acids at positions specified by an amber (UAG) codon in the lacI mRNA. The resulting set of over 4000 single amino acid replacements in the lac repressor protein allows a detailed analysis of its substitution tolerance along the linear array of residues, and reveals structure-function relationships in lac repressor and in proteins in general. (1) There are two main regions in the repressor which are extremely sensitive to amino acid replacements. One, the amino-terminal 59 residues, has been implicated in DNA and operator binding by a large body of work. The second, extending from approximately residues 239 to 289/292, forms the repressor core and shares the most homology with other repressor and DNA binding proteins. (2) Throughout the rest of the protein, segments of 6 to 14 amino acids, which are highly tolerant to single amino acid replacements, appear to act as "spacers" between one or several hydrophobic residues that are relatively intolerant to substitutions. (3) We have replaced the amino acids in these tolerant regions with spans of alanine residues, from 5 to 13 amino acids. In all five of the regions tested, alanine replacements, sometimes of up to 8 amino acids, still allowed functional repressor, while deletion of the same residues destroyed repressor function. This reinforces the view that many regions of a protein do not require a specific sequence to serve as spacers between more important residues. (4) A distinct pattern of substitutions leading to the I(s) phenotype suggests the location of residues involved in inducer binding. (5) A number of general substitution patterns can be recognized. For instance, proline is not tolerated at over 40 sites which tolerate all the other amino acid replacements. Another set of sites tolerates only non-polar amino acids, whereas a third set tolerates a subset of the smallest amino acids, (serine, alanine, glycine and cysteine, and sometimes threonine and valine). (5) Overall, 93 of 328 sites (28%) tolerate all 13 amino acids tested, and 144 of 328 (44%) tolerate 12/13 or all 13 substitutions. We judge that 192 of 328 sites (59%) are generally tolerant to substitutions.
J Mol Biol 1994 Jul 29
PMID:Genetic studies of the lac repressor. XIV. Analysis of 4000 altered Escherichia coli lac repressors reveals essential and non-essential residues, as well as "spacers" which do not require a specific sequence. 804 48

The pathway for glycerol catabolism in Streptomyces coelicolor is determined by the gylCABX operon, which is transcribed from two closely spaced glycerol-inducible, glucose-repressible promoters. Glucose (or catabolite) repression of gyl is known to be exerted by a general catabolite repression system in which the soluble glucose kinase plays a central role. The gylR gene is contained in a separate glycerol-inducible, weakly glucose-repressible transcription unit immediately upstream from the gyl operon. The role of gylR in the regulation of gyl transcription was assessed by introducing specific null mutations into the chromosomal gylR gene. Direct quantification of gyl transcripts from the gylR null mutants grown on different carbon sources demonstrated that GylR is the repressor of the gylCABX operon and also revealed that GylR functions as a negative autoregulator. Moreover, the transcriptional analysis revealed that the gylR null mutants were relieved of glucose repression of both gylCABX and gylR. We conclude that both substrate induction and catabolite repression of gyl are mediated through the GylR protein. This is the first direct evidence that catabolite repression in Streptomyces is not exerted at the transcriptional level by a general 'catabolite repressor protein'. Models for catabolite repression are discussed.
Mol Microbiol 1994 Jun
PMID:Substrate induction and catabolite repression of the Streptomyces coelicolor glycerol operon are mediated through the GylR protein. 805 26

The expression regulation of spvR, a regulatory gene on the virulence plasmid (pKDSC50) of Salmonella choleraesuis serovar Choleraesuis, was investigated by spvR-lacZ translational fusion. The spvR gene was found to be positively regulated by its own product, the SpvR protein, and this unusual positive autoregulation was repressed by the products of spvA and spvB, virulence-associated genes present downstream from the spvR gene. Amino acid sequence analysis revealed that the amino-terminal region of SpvB had homology with the CatM repressor protein of Acinetobacter calcoaceticus, which belongs to the MetR/LysR protein family. On the other hand, the sigma factor RpoS was required for expression of the spvR gene in the stationary phase of bacterial growth. The SpvR protein was also necessary for self-activation, suggesting that an RNA polymerase holoenzyme containing RpoS requires SpvR protein in order to recognize the spvR promoter.
Mol Microbiol 1994 Jun
PMID:Regulation of spvR gene expression of Salmonella virulence plasmid pKDSC50 in Salmonella choleraesuis serovar Choleraesuis. 805 29

Using the catechol dehydrogenase gene as a reporter, we isolated random mutations in the plJ101 korB gene operator/promoter (OP) region that affect korB expression and regulation. We mapped these mutations to inverted repeat sequences within the promoter and studied their effects on binding of the KorB repressor protein to the OP, on expression of the korB gene, and on plasmid transmission during mating. Additionally, we investigated the biological effects of KorB binding to a locus (sti, for strong incompatibility) adjacent to the korB OP and implicated in plJ101 replication. Our results identify sites that influence the synthesis and autoregulation of KorB; they also show that interaction of KorB with sti affects repression of korB and transmission of plasmids to spores of recipients.
Mol Microbiol 1994 Apr
PMID:Mutations that affect regulation of the korB gene of Streptomyces lividans plasmid plJ101 alter plasmid transmission. 805 38


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>