Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three temperature-sensitive mutant strains for RNA polymerase beta or beta' subunits (carrying mutations tsx, A2R7 and R120) were used in order to investigate the dependence of the induced lac expression on stimulation by cyclic AMP after the shift to non-permissive temperature. High temperature lowered the rate of beta-galactosidase synthesis. However, the low rate of synthesis could be strongly increased by cyclic AMP (30, 2.4 and 5.7-fold increases for tsX, A2R7 and R120 mutants, respectively). At the permissive temperature stimulation by cyclic AMP was less than 1.4-fold (minimal medium supplemented with glycerol). The results suggest that the maximal expression of the lac operon is saturated, that is, a hypothetical increase in RNA polymerase or cAMP-CRP concentration in the cell with not enhance the expression. The concept of saturation explains why it was possible to increase the beta-galactosidase synthesis in conditions of limited promoter binding activity of RNA polymerase through increase in concentration of cyclic AMP-CRP complex in the cell (addition of cyclic AMP) to the values higher than that observed on glycerol.
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PMID:Expression of the lac operon in RNA polymerase mutants of Escherichia coli K12. 22 41

The dnaJ deletion mutant K7052(lambda dnaK) has a temperature-sensitive defect in the synthesis of beta-galactosidase. We confirmed this operon-specific and temperature-sensitive defect in cell-free extracts prepared from the mutant cells and found that the missing factor was CRP. In the mutant, the cellular concentration of CRP was too low to allow the expression of the lac operon at a nonpermissive temperature. Introduction of a CRP over-producing plasmid into the dnaJ deletion mutant suppressed the defect of beta-galactosidase synthesis. The lower content of CRP in the mutant was found to result from extreme instability of the protein. These results strongly suggested that the heat shock protein dnaJ is involved in the stabilization (or degradation) of CRP.
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PMID:Escherichia coli dnaJ deletion mutation results in loss of stability of a positive regulator, CRP. 161 21

The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced. A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ). The operon is transcribed in the order lacEFGZK. The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A. radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon). The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.
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PMID:Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter. 163 Mar 15

The glpE gene of E. coli was found to be transcribed divergently with respect to glpD, which is adjacent to glpE head-to-head on the E. coli chromosome. We constructed glpD- and/or glpE-lacZ fusion plasmids, which provided glpD and lacZ as reporter genes. The expression of glpD and glpE, under the control of the cAMP-CRP complex, was examined by measuring the activities in E. coli cells of beta-galactosidase encoded by lacZ and glycerol-3-phosphate dehydrogenase encoded by glpD. In the double-reporter-gene system, the expression of glpD and glpE was found to be positively regulated by cAMP-CRP. We also confirmed that intracellular levels of the translation products and the transcripts from glpD and glpE were positively regulated by cAMP-CRP. The cAMP-mediated induction of gene expression of glpD and glpE was significantly affected by structural alterations of the single CRP-binding site between glpD and glpE. These results indicate that the single CRP-binding site is a cis-acting element involved in the positive regulation of the expression of both glpD and glpE at the transcriptional level.
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PMID:Regulation of glpD and glpE gene expression by a cyclic AMP-cAMP receptor protein (cAMP-CRP) complex in Escherichia coli. 184 66

Measurements of cyclic phosphodiesterase, or of beta-galactosidase in the case of cpdB'-'lacZ fusions, indicate that cpdB expression in both Escherichia coli and Salmonella typhimurium is modulated by carbon source availability, consistent with previous observations in Salmonella. Nucleotide sequence analysis and transcription mapping of both cpdB genes have revealed, in their 5' flanking regions, sequences with good similarity to consensus -10 and -35 regions and cyclic AMP-cyclic AMP receptor protein (cAMP-CRP) binding sites. Furthermore, they are strongly conserved in both organisms. Deletion analysis of an E. coli cpdB'-'lacZ fusion supports the identification of these elements, and a role for the cAMP-CRP binding site in modulating constitutive cyclic phosphodiesterase expression.
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PMID:Transcription and regulation of the cpdB gene in Escherichia coli K12 and Salmonella typhimurium LT2: evidence for modulation of constitutive promoters by cyclic AMP-CRP complex. 217 62

A plasmid carrying a CRP-dependent promoter fused to the lac structural genes was manipulated to construct a set of spacing mutants that have varying lengths between the CRP binding site and the -35 region. The lengths of the spacer were changed over 45 bp by inserting or deleting nucleotides. DNase I footprinting analysis revealed that the spacer length did not affect the binding of cAMP-CRP to the CRP site. The effect of the spacer length on transcription activation by cAMP-CRP was tested in vivo by beta-galactosidase and quantitative S1 assays with crp+ and delta crp cells harboring plasmids. Insertions or deletions of non-integral helical turns, which displace the CRP site onto the opposite face of DNA helix compared to the original promoter, eliminated completely the activation of transcription. In contrast, changing the spacer length by integral helical turns allowed the promoter to respond to CRP, although the degree of activation varied with the length of the spacer. We conclude that stereospecific positioning of CRP and RNA polymerase on the DNA helix is strictly required for CRP action. The data support a model that CRP stimulates transcription by directly contacting RNA polymerase.
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PMID:Helical phase dependent action of CRP: effect of the distance between the CRP site and the -35 region on promoter activity. 217 26

On the basis of Escherichia coli DNA and vectors pBR322, pUC19, hybrid plasmids restoring Udp+ phenotype in the E. coli deletion (delta udp) mutant have been obtained. The udp gene is carried by a 8 kb PstI fragment (on the pUD2) and by a smaller 2.87 kb PstI-SalGI fragment from the PstI fragment (pUD7). The uridine phosphorylase level was 30 times higher in the cells containing hybrid plasmid as compared to the strain with chromosomal location of the udp gene. On the other hand, the measurements of uridine phosphorylase activity in the cytR- and cya- background indicate that expression of the cloned udp gene escapes partially negative control of the CytR repressor and positive control of cAMP--CRP complex. These data suggest that the 2.87 kb PstI--SalGI-fragment contains the intact udp gene which is transcribed from its own promoter. Increase in the activity of beta-galactosidase encoded by udp-lacZ fusion has been observed in the presence of pUD2 or pUD7, which was suggested to be the consequence of titration of CytR repressor molecules in the operator region of the cloned udp.
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PMID:[Cloning the uridine phosphorylase (udp) gene of Escherichia coli and its expression in recombinant plasmids]. 251 86

Analysis of the induction of expression of cea-lacZ fusions in cya and crp mutants showed that catabolite repression affects the kinetics of induction and the rate of induced synthesis. In a cya mutant, addition of cAMP reduced the induction lag and increased the amount of beta-galactosidase produced. The CRP-cAMP complex was found to bind to two sites 5' to the cea promoter, but deletion analysis showed that only one of these was involved in the control of cea. Deletion of this site resulted in a loss of the stimulatory effects of cAMP in a cya mutant.
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PMID:Interaction of the CRP-cAMP complex with the cea regulatory region. 254 Apr 17

Interaction of negative (CytR) and positive (cAMP-CRP) control in the promoter region of the uridine phosphorylase (udp) gene of Escherichia coli has been studied by using udp-lac operon fusions in which the structural lacZ gene is expressed from the wild type promoter udpP+ or from mutant promoters udpP1 and udpP18. The specific activity of beta-galactosidase was examined in these fusions in cytR+ and cytR- backgrounds after introduction of specific mutations in crp locus, crp* and crp(a) altering interaction of CRP protein with catabolite-sensitive promoters. The data obtained using crp* mutation confirm the proposed model of the udp gene regulation, according to which CytR repressor protein interferes with CRP binding site in the promoter-operator region of the udp gene and thereby prevents the positive action of cAMP-CRP complex on the udp expression. Additional data in favor of this model were obtained using crp(a) mutation which most probably alters the structure of CRP protein in such a way that it exhibits more high affinity to the udp promoter, as compared to the CytR repressor protein. Indeed, taken by itself, the crp(a) mutation did not lead to any increase in the expression of udpP+-lac fusion under the conditions of cAMP limitation (on glucose-grown cells), in spite of whether or not the CytR repressor was present. However, when combined with the ptsG mutation or when cells were grown on succinate medium, complete constitutive expression of udpP+-lac fusion is observed, even in the presence of the cytR gene product. The effect of the crp(a) mutation was virtually the same in strains harboring udpP1-lac fusion. These data are in accordance with suggestion that udpP1 is a mutation in the site of the promoter-operator region that responds to the cytR gene product, while the corresponding binding site for CRP protein is still unaltered in this mutant. On the other hand, the crp(a) mutation causes only slight alteration in the expression of udpP18-lac fusion, providing additional evidence that udpP18 mutation seems to comprise a modification of the promoter-operator region, where binding sites for CRP and CytR proteins overlap.
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PMID:[Interaction of negative (CytT) and positive (cAMP-CRP) regulation in the promoter region of the uridine phosphorylase (udp) gene in Escherichia coli K-12]. 266 21

Mutations which permit cAMP binding protein (CRP) to act in the absence of cAMP have been isolated by in vitro mutagenesis of a plasmid containing the cloned crp gene. Adenylate cyclase deficient cells harbouring the mutant (crp*) plasmids exhibited a variety of fermentation profiles on MacConkey indicator plates containing various sugars. beta-galactosidase synthesis in cells carrying the crp* plasmids was activated most by the addition of cGMP as well as cAMP. The sites of mutations which are responsible for the cAMP independent phenotype were determined by in vitro recombination and DNA sequencing. The amino acid substitutions in the mutant proteins were found in two specific regions of the crp gene encoding residues 53-62 and 141-148 of CRP polypeptide. The first region may participate in cAMP binding, while the second appears to be the inter-domain region of the N-terminal cAMP-binding and C-terminal DNA-binding domains.
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PMID:Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli. 300 51


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