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Results for “Cyclic AMP Receptor Protein”

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At least 19 recordsLinked to original sources
PubMed1977

Effects of cyclic nucleotides on the conformational states of the alpha core of the cyclic AMP receptor protein.

The alpha core gragment produced by limited proteolysis contains the cyclic AMP binding domain and the two buried sulfhydryl groups of the cyclic AMP receptor protein. The buried sulfhydryl groups of the alpha core react with 5,5'-dithio-bis(2-nitrobenzoic acid) after denaturation by 3 M urea or digestion with subtilisin. The rate of sulfhydryl modification in the presence of 3 M urea or subtilisin is markedly decreased in the presence of cyclic nucleotides which are proposed to tighten the conformation of the alpha core. Incubation of the alpha core in 3 M urea or dithionitrobenzoic acid does not affect cyclic AMP binding while dithionitrobenzoic acid plus 3 M urea inhibits cyclic AMP binding suggesting a role for the buried sulfhydryls in cyclic AMP binding or their proximity to the cyclic AMP binding domain of the alpha core. The data are consistent with a ligand-induced conformational change in the alpha region of the native cyclic AMP receptor protein that is required for DNA binding.

Binding Sites↗
PubMed1979

Presence of free cyclic AMP receptor protein and regulation of its level by cyclic AMP in neuroblastoma-glioma hybrid cells.

Neuroblastoma-glioma hybrid cells of line 108CC-5 were found to contain high levels of soluble adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase activity and high levels of two specific cAMP receptor proteins, RI and RII. Treatment of the hybrid cells with dibutyryl cAMP increased the level of RI but did not significantly affect the level either of RII or of cAMP-dependent protein kinase activity. The effect of dibutyryl cAMP could be mimicked by prostaglandin E1 and 3-isobutyl-1-methylxanthine, both of which are known to raise cAMP levels in neuroblastoma-glioma hybrid cells. Both in control as well as in dibutyryl cAMP-treated cells, RII but not RI was associated with cAMP-dependent protein kinase. Several lines of evidence suggest that RI represents the free regulatory subunit of type I cAMP-dependent protein kinase. The presence of this regulatory subunit as free cAMP receptor protein in neuroblastoma-glioma hybrid cells may be of significance with respect to the regulation of growth and differentiation in tumor cells.

Animals↗
PubMed1978

A new extragenic suppressor of cya mutation. Mutant cyclic AMP receptor protein with an increased affinity for cyclic AMP.

A strain bearing an extragenic suppressor of cya mutation was isolated as a second-site revertant of an adenylate cyclase deficient strain. The mutant was unable to synthesize cAMP but showed normal fermentation profiles and growth properties on a variety of carbon sources. The site of reversion was mapped in, or near, the structural gene for the cAMP receptor protein. Structural alteration of the protein was directly demonstrated by the following biochemical observations: (i) A 10-fold decrease in the dissociation constant for cAMP, (ii) an acidic shift in the isoelectric point, and (iii) the altered binding properties to lambdah80dlac ps DNA.

Carbohydrate Metabolism↗
PubMed1979

Interaction site of Escherichia coli cyclic AMP receptor protein on DNA of galactose operon promoters.

Cyclic AMP (cAMP) and its receptor protein (CRP) have a dual role in the regulation of the two promoters that control the galactose (gal) operon of Escherichia coli. One promoter, P1, requires cAMP-CRP for activity; the other, P2, is inhibited by these factors. We have examined the interactions site of cAMP-CRP on gal DNA by using two types of protection experiments, involving DNase digestion and methylation by dimethyl sulfate. Our results indicate that cAMP-CRP binds to gal DNA in a segment located between 50 and 24 base pairs preceding the P1 start point for transcription. Although the location of the cAMP-CRP interaction site is clearly different in gal and lac DNA, comparison of the DNA sequences suggests a similar recognition sequence. The location of the cAMP . CRP-binding site in gal further suggests that protein-protein interactions between RNA polymerase and cAMP . CRP play an important role in transcription initiation at the gal and possibly other cAMP-dependent promoters.

Base Sequence↗
PubMed1978

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein.

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.

Aerobiosis↗
PubMed1976

The use of affinity chromatography in purification of cyclic nucleotide receptor proteins.

Biospecific affinity chromatography has been used to purify specific cyclic AMP and cyclic GMP receptor proteins. Several variables are important for successful purification of the cyclic AMP receptor protein, the most critical being the length of the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to agarose specifically retains the cyclic AMP receptor protein by interaction with the immobilized nucleotide. Binding of the cyclic AMP receptor subunit of cyclic AMP-dependent protein kinase to the immobilized nucleotide results in dissociation of the catalytic protein phosphokinase subunit which is not retained. The retained cyclic AMP receptor protein is subsequently eluted by cyclic AMP. Homogeneous cyclic AMP receptor protein prepared from rabbit skeletal muscle by affinity chromatography has been characterized. The molecular weight of the native protein as determined by analytical ultracentrifugation and polyacrylamide gel electrophoresis at varying acrylamide concentrations is 76 800 and 82 000, respectively. The protein is asymmetric with frictional and axial ratios of 1.64 and 12. SDS and urea polyacrylamide gel electrophoresis indicate that the native cyclic AMP receptor is composed of two identical subunits of 42 700 molecular weight. The native protein dimer binds 2 moles of cyclic AMP per mole of protein and is active in suppressing activity of isolated catalytic subunits of cyclic AMP-dependent protein kinase. Cyclic GMP receptor protein from bovine lung has been purified using the same affinity chromatography media. Since cyclic nucleotide binding to cyclic GMP-dependent protein kinase does not result in dissociation of regulatory receptor and catalytic phosphotransferase subunits, the cyclic GMP-dependent protein kinase holoenzyme is retained on the column and can be subsequently specifically eluted with cyclic GMP.

Animals↗
PubMed1977

Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand.

Two protein bands, present in cytosol fractions from each of seven rat tissues examined, specifically incorporated 32P-labeled 8-azidoadenosine 3':5'-monophosphate (8-N3-[32P]cAMP), a photoaffinity label for cAMP-binding sites. These proteins had apparent molecular weights of 47,000 and 54,000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. These two proteins were characterized in three of the tissues, namely, heart, uterus, and liver, by the total amount of 8-N3-[32P]cAMP incorporation, by the dissociation constant (Kd) for 8-N3-[32P]cAMP, and by the nucleotide specific inhibition of 8-N3-[32P]cAMP incorporation. Several lines of evidence were obtained that the protein with an apparent molecular weight of 47,000 represents the regulatory subunit of a type I cAMP-dependent protein kinase, while the protein with an apparent molecular weight of 54,000 represents the regulatory subunit of a type II cAMP-dependent protein kinase. Almost all of the cAMP receptor protein found in the cytosol of these tissues, as measured by 8-N3-[32P]cAMP incorporation, was associated with these two protein kinases, in agreement with the idea that most effects of cAMP are mediated through protein kinases. The photoaffinity labeling with 8-N3-[32P]cAMP can be used to estimate quantitatively the amounts of regulatory subunit of type I and type II cAMP-dependent protein kinases in various tissues.

Affinity Labels↗
PubMed2025

Genome-wide mapping of cyclic AMP receptor protein binding in Enteroaggregative Escherichia coli reveals targeting of virulence-associated genes.

Bacterial pathogens use a wide array of virulence factors to colonise and subsequently elicit disease in their host. These factors are often subject to extensive regulation at the transcriptional level, to ensure that their expression is timely. Although many pathogens use bespoke transcription factors that primarily target virulence genes, global transcription factors also sometimes play a role in controlling these genes. Enteroaggregative Escherichia coli (EAEC) is a significant cause of watery and mucoid diarrhoea globally. The organism colonises the small intestine before producing toxins that elicit disease, using a multitude of virulence factors that are encoded both chromosomally and on virulence plasmids. In this work, we have studied the cAMP Receptor Protein (CRP), a well-characterised bacterial global transcription factor, focusing on its role in pathogenicity of the prototype EAEC strain 042. We show that, although most functional CRP binding sites on the chromosome are conserved between E. coli K-12 and 042, CRP has been co-opted to couple the expression of some virulence genes to the nutritional state of the cell. We report novel mechanisms for CRP-dependent regulation of genes, whose products contribute to adhesion, production of a bacterial antibiotic, and export of a polysaccharide capsule.

CRP↗
PubMed1978

DNA sequence of the araBAD promoter in Escherichia coli B/r.

The L-arabinose operon in Escherichia coli is a model system for the study of the control of gene expression. Maximal expression of the araBAD operon requires two positive control components: the araC protein-L-arabinose complex and the cyclic AMP receptor protein-cyclic AMP complex. Both araC protein and cyclic AMP receptor protein are required for the initiation of transcription of araBAD mRNA. We have used the plasmid pBR322 as a vector for cloning DNA fragments that contain the araBAD promoter. The cloned ara fragments were identified by both physical and genetic tests. A restriction map was constructed and the DNA sequence of the promoter was determined. The promoter contains a site that is similar to the RNA polymerase recognition sites in the galactose and lactose operons. It also contains a region similar to the known cyclic AMP receptor protein binding sites in the galactose and lactose operons.

Arabinose↗
PubMed1976

Effects of crp mutations on adenosine 3',5'-monophosphate metabolism in Salmonella typhimurium.

Wild-type Salmonella typhimurium could not grow with exogenous cyclic adenosine 3',5'-monophosphate (AMP) as the sole source of phosphate, but mutants capable of cyclic AMP utilization could be isolated provided the parental strain contained a functional cyclic AMP phosphodiesterase.All cyclic AMP-utilizing mutants had the growth and fermentation properties of cyclic AMP receptor protein (crp) mutants, and some lacked cyclic AMP binding activity in vitro. The genetic defect in each such mutant was due to a single point mutation, which was co-transducible with cysG. crp mutants isolated by alternative procedures also exhibited the capacity to utilize cyclic AMP. crp mutants synthesized cyclic AMP at increased rates and contained enhanced cellular cyclic AMP levels relative to the parental strains, regardless of whether or not cyclic AMP phosphodiesterase was active. Moreover, adenylate cyclase activity in vivo was less sensitive to regulation by glucose, possibly because the enzyme II complexes of the phosphotransferase system, responsible for glucose transport and phosphorylation, could not be induced to maximal levels. This possibility was strengthened by the observation that enzyme II activity (measured both in vitro by sugar phosphorylation and in vivo by sugar transport and chemotaxis) was inducible in the parental strain but not in crp mutants. The results suggest that the cyclic AMP receptor protein regulates cyclic AMP metabolism as well as catabolic enzyme synthesis.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Cyclic AMP receptor protein-dependent activation of the Escherichia coli acsP2 promoter by a synergistic class III mechanism.

The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by "focusing" RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions -69.5 and -122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the alpha subunit of RNA polymerase holoenzyme (alpha-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the alpha-CTD. Other surface-exposed residues in the alpha-CTD contributed to acs transcription, suggesting that the alpha-CTD may interact with at least one protein other than CRP.

Acetate-CoA Ligase↗
PubMed1979

Cyclic AMP receptor triggers nuclear protein phosphorylation in a hormone-dependent mammary tumor cell-free system.

Adenosine 3',5'-monophosphate (cyclic AMP) receptor protein of 56,000 daltons increases markedly in mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) after incubation of tumor slices with cyclic AMP, benzamide, and arginine. Incubation of cytosol from these tumor slices with nuclei from unincubated tumors results in nuclear uptake of the 56,000-dalton cyclic AMP receptor and in phosphorylation of the 76,000-dalton nuclear protein. Binding of the 56,000-dalton receptor and phosphorylation of the 76,000-dalton protein also occur in DMBA tumor nuclei when protein kinase type II of bovine heart is used. The results suggest that cyclic AMP receptor is involved in the nuclear events of a hormone-dependent mammary tumor.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1975

Cyclic adenosine monophosphate receptor: effect of cyclic AMP analogues on DNA binding and proteolytic inactivation.

The cyclic AMP receptor protein of Escherichia coli in the presence of cyclic AMP undergoes a conformational change resulting in an increased affinity for DNA and an increased susceptibility to attack by proteolytic enzymes resulting in loss of DNA binding capacity. Of several cyclic nucleotides tested only cyclic AMP and cyclic tubercidin monophosphate are able to effect the conformational transition in cyclic AMP receptor protein, prerequisite to proteolytic inactivation or DNA binding. Other analogues such as cyclic GMP or cyclic IMP which are competitive inhibitors of cyclic AMP do not support DNA binding or proteolytic inactivation.

Bacterial Proteins↗
PubMed1979

Regulation of synthesis of a major outer membrane protein: cyclic AMP represses Escherichia coli protein III synthesis.

Cyclic AMP is the effector molecule for both positive and negative control of synthesis of several Escherichia coli proteins. Among the latter is the major outer membrane protein III. The control mechanism occurs at the level of transcription and involves the cyclic AMP receptor protein. The repressing system is saturated at lower concentrations of cyclic AMP than is the positive control system.

Bacterial Proteins↗