Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

ABSTRACT: Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

SUBMITTER: Pinkney M

PROVIDER: S-EPMC1148940 | biostudies-other | 1988 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Mutations in the cyclic AMP binding site of the cyclic AMP receptor protein of Escherichia coli.

Project description:Mutants in the cyclic AMP binding site of the cyclic AMP receptor protein (CRP) of Escherichia coli have been constructed by oligonucleotide-directed mutagenesis. They have been phenotypically characterized and their ability to enhance the expression of catabolite-repressible operons has been tested. In addition, the binding of cyclic nucleotides to the mutants has been investigated. It is shown that the six mutants made fall into one of three classes: (i) those that bind cyclic AMP better than the wild type protein (Ser-62----Ala) and result in greater transcription enhancement; (ii) those that bind cyclic AMP similarly to wild type (Ser-83----Ala, Ser-83----Lys, Thr-127----Ala, Ser-129----Ala); and (iii) those that do not bind cyclic AMP at all (Arg-82----Leu). Implications of these findings with respect to present models of the cyclic nucleotide binding pocket of CRP are discussed.

| S-EPMC1149374 | biostudies-other

Transcription activation at Escherichia coli promoters dependent on the cyclic AMP receptor protein: effects of binding sequences for the RNA polymerase alpha-subunit.

Project description:Transcription activation at two semi-synthetic Escherichia coli promoters, CC(-41.5) and CC(-72.5), is dependent on the cyclic AMP receptor protein (CRP) that binds to sites centred 41.5 and 72.5 bp upstream from the respective transcription startpoints. An UP-element that can bind the C-terminal domain of the RNA polymerase (RNAP) alpha-subunit was cloned upstream of the DNA site for CRP at CC(-41.5) and downstream of the DNA site for CRP at CC(-72.5). In both cases CRP-dependent promoter activity was increased by the UP-element, but CRP-independent activity was not increased. DNase I footprinting was exploited to investigate the juxtaposition of bound CRP and RNAP alpha-subunits. In both cases, CRP and RNAP alpha-subunits occupy their cognate binding sites in ternary CRP-RNAP promoter complexes. RNAP alpha-subunits can occupy the UP-element in the absence of CRP, but this is not sufficient for open complex formation. The positive effects of binding RNAP alpha-subunits upstream of the DNA site for CRP at -41.5 are suppressed if the UP-element is incorrectly positioned.

| S-EPMC1135802 | biostudies-other

The cyclic AMP-cyclic AMP receptor protein complex regulates activity of the traJ promoter of the Escherichia coli conjugative plasmid pRK100.

Project description:The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of traJ expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ-lacZ reporter system. A comparison of the enzyme activities in the wild-type Escherichia coli strain MC4100 with those in cya and crp mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the traJ promoter of the F-like plasmid pRK100. The defect in the cya mutant was partially restored by addition of exogenous cAMP. Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription. Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the traJ promoter region. DNase I footprint experiments mapped the CRP binding site around position -67.5 upstream of the putative traJ promoter. Targeted mutagenesis of the traJ promoter region confirmed the location of the CRP binding site. Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in cya or crp exhibited reduced conjugal transfer from pRK100.

| S-EPMC148056 | biostudies-literature

The tib adherence locus of enterotoxigenic Escherichia coli is regulated by cyclic AMP receptor protein.

Project description:Enterotoxigenic Escherichia coli (ETEC) is a Gram-negative enteric pathogen that causes profuse watery diarrhea through the elaboration of heat-labile and/or heat-stable toxins. Virulence is also dependent upon the expression of adhesive pili and afimbrial adhesins that allow the pathogen to adhere to the intestinal epithelium or mucosa. Both types of enterotoxins are regulated at the level of transcription by cyclic AMP (cAMP) receptor protein (CRP). To further our understanding of virulence gene regulation, an in silico approach was used to identify putative CRP binding sites in the genome of H10407 (O78:H11), an ETEC strain that was originally isolated from the stool of a Bangledeshi patient with cholera-like symptoms circa 1971. One of the predicted binding sites was located within an intergenic region upstream of tibDBCA. TibA is an autotransporter and afimbrial adhesin that is glycosylated by TibC. Expression of the TibA glycoprotein was abolished in an H10407 crp mutant and restored when crp was provided in trans. TibA-dependent aggregation was also abolished in a cyaA::kan strain and restored by addition of exogenous cAMP to the growth medium. DNase I footprinting confirmed that the predicted site upstream of tibDBCA is bound by CRP. Point mutations within the CRP binding site were found to abolish or significantly impair CRP-dependent activation of the tibDB promoter. Thus, these studies demonstrate that CRP positively regulates the expression of the glycosylated afimbrial adhesin TibA through occupancy of a binding site within tibDBp.

| S-EPMC3067628 | biostudies-literature

Novel Escherichia coli RNA Polymerase Binding Protein Encoded by Bacteriophage T5.

Project description:The Escherichia coli bacteriophage T5 has three temporal classes of genes (pre-early, early, and late). All three classes are transcribed by host RNA polymerase (RNAP) containing the ?70 promoter specificity subunit. Molecular mechanisms responsible for the switching of viral transcription from one class to another remain unknown. Here, we find the product of T5 gene 026 (gpT5.026) in RNAP preparations purified from T5-infected cells and demonstrate in vitro its tight binding to E. coli RNAP. While proteins homologous to gpT5.026 are encoded by all T5-related phages, no similarities to proteins with known functions can be detected. GpT5.026 binds to two regions of the RNAP ? subunit and moderately inhibits RNAP interaction with the discriminator region of ?70-dependent promoters. A T5 mutant with disrupted gene 026 is viable, but the host cell lysis phase is prolongated and fewer virus particles are produced. During the mutant phage infection, the number of early transcripts increases, whereas the number of late transcripts decreases. We propose that gpT5.026 is part of the regulatory cascade that orchestrates a switch from early to late bacteriophage T5 transcription.

| S-EPMC7472727 | biostudies-literature

19F n.m.r. studies of conformational changes accompanying cyclic AMP binding to 3-fluorophenylalanine-containing cyclic AMP receptor protein from Escherichia coli.

Project description:A fluorine-containing analogue of the cyclic AMP (cAMP) receptor protein (CRP) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorophenylalanine (3-F-Phe). 19F n.m.r. studies on this protein have provided direct evidence for cAMP-induced conformational changes not only within the cAMP-binding domain but also within the hinge region connecting the cAMP-binding domain to the DNA-binding headpiece. At 313 K, the 19F n.m.r. spectrum of [3-F-Phe]CRP showed five signals corresponding to the five phenylalanine residues as expected for a symmetrical dimer. Proteolysis of [3-F-Phe]CRP with subtilisin produced a fragment (the alpha-fragment) containing the cAMP-binding domain. The alpha-fragment contains all the phenylalanines except for Phe-136, a residue located in the hinge region. By comparing the 19F spectra of [3-F-Phe]CRP and its alpha-fragment, the signal for Phe-136 was assigned. The chemical shifts of the corresponding signals in the two spectra are similar, indicating that the alpha-fragment retains the structure it has in the intact protein. The largest cAMP-induced shift was observed for the signal from Phe-136 providing direct evidence for a conformational change in the hinge region. However, whereas binding of a single cAMP molecule to a CRP dimer is known to be sufficient to activate the DNA binding, the n.m.r. data indicate that the hinge region does not have the same conformation in both subunits when only one cAMP molecule is bound.

| S-EPMC1133211 | biostudies-other

Genome-wide identification of transcription start sites yields a novel thermosensing RNA and new cyclic AMP receptor protein-regulated genes in Escherichia coli.

Project description:Intergenic regions often contain regulatory elements that control the expression of flanking genes. Using a deep-sequencing approach, we identified numerous new transcription start sites in Escherichia coli, yielding a new thermosensing regulatory RNA and seven genes previously unknown to be under the control of the global regulator CRP.

| S-EPMC3133129 | biostudies-literature

Binding of the Escherichia coli cyclic AMP receptor protein to DNA fragments containing consensus nucleotide sequences.

Project description:Binding of the Escherichia coli CRP protein to DNA fragments carrying nucleotide sequences closely corresponding to the consensus is very tight with a dissociation time of over 2 h in our conditions. The concentration of cyclic AMP required for this binding is below the physiological range of intracellular cyclic AMP concentrations. Changes in nucleotide sequence at positions that are not well-conserved between different naturally-occurring CRP sites allow a more rapid dissociation of CRP-DNA complexes. There is an inverse correlation between the stability of CRP binding to sites in vitro and the repression by glucose of expression dependent on these sites in vivo: expression that is dependent on the tighter binding sites cannot be repressed by the inclusion of glucose in the growth medium.

| S-EPMC1138872 | biostudies-other

Activation of sigma 28-dependent transcription in Escherichia coli by the cyclic AMP receptor protein requires an unusual promoter organization.

Project description:The Escherichia coli aer regulatory region contains a single promoter that is recognized by RNA polymerase containing the flagellar sigma factor, sigma(28). Expression from this promoter is dependent on direct activation by the cyclic AMP receptor protein, which binds to a target centred 49.5 base pairs upstream from the transcript start. Activator-dependent transcription from the aer promoter was reconstituted in vitro, and a tethered inorganic nuclease was used to find the position of the C-terminal domains of the RNA polymerase alpha subunits in transcriptionally competent open complexes. We report that the ternary activator--RNA polymerase--aer promoter open complex is organized differently from complexes at previously characterized promoters. Among other E. coli promoters recognized by RNA polymerase containing sigma(28), only the trg promoter is activated directly by the cyclic AMP receptor protein. The organization of the different promoter elements and the activator binding site at the trg promoter is the same as at the aer promoter, suggesting a common activation mechanism.

| S-EPMC2859248 | biostudies-literature

ChIP-seq of the cyclic-AMP Receptor Protein (CRP) and RNA polymerase sigma 70 subunit in Vibrio cholerae

Project description:The experiment contains ChIP-seq data for Vibrio cholerae strain N16961. The strain was grown at 37 degrees in M9 minimal medium and crosslinked with 1 % (v/v) formaldehyde. After sonication, to break open cells and fragment DNA, immunoprecipitations were done using no antibodies (mock immunoprecipitations) or antibodies against CRP or the RNA polymerase sigma 70 subunit. Libraries were prepared using DNA remaining after immunoprecipitation.

2019-02-04 | E-MTAB-6472 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data