cAMP Receptor Protein Binding Research Articles

Molecular and functional characterization of a carbon starvation gene of Escherichia coli

Escherichia coli induces the synthesis of at least 30 proteins at the onset of carbon starvation, two-thirds of which are positively regulated by the cyclic AMP (cAMP) and cAMP receptor protein (CRP) complex. Two of the cAMP-CRP-dependent genes mapped to 14 and 93 minutes of the chromosome and are designated cstA and cstB, respectively. The cstA promoter region was cloned and localized to a 600 base-pair fragment downstream from the iron-regulated entCEBA-P15 operon. Carbon starvation-inducible transcription initiated at three sites spaced one turn of the DNA helix apart. All had −10 sequences similar to consensus Eσ 70 promoters and poor −35 sequences. Deletion of a putative CRP binding site abolished carbon starvation-mediated induction. Sequence analysis of the cstA coding region revealed the presence of three sequential open reading frames potentially encoding two hydrophobic proteins of 60,223 Da and 15,201 Da and a hydrophilic protein of 7467 Da. Overexpression of the cstA region produced starvation-inducible proteins of the expected sizes. Suggestive evidence was obtained that cstA is involved in peptide utilization.

Three adjacent binding sites for cAMP receptor protein are involved in the activation of the divergent malEp-malKp promoters.

The divergent malEFG and malK-lamB-malM operons in Escherichia coli are controlled by partially overlapping promoters, whose activity depends on the presence of two transcriptional activators, MalT and the cAMP receptor protein (CRP). The 271-base-pair region separating the transcription start points of the promoters malEp and malKp comprises a compact array of binding sites for MalT and CRP. We report the characterization of the in vitro interactions of CRP with its four adjacent binding sites and the analysis of their function in vivo. By using the DNase I footprinting technique, we showed that CRP binds with high affinity to the three malEp-proximal sites and with a low affinity to the fourth site. CRP binding to these sites is not cooperative, even though they are adjacent and located on the same face of the DNA double helix. Each of these sites was destroyed by localized mutagenesis and the residual activity of the promoters was measured in vivo. Mutations in any of the three high-affinity binding sites reduced both malEp and malKp activity. The participation of several adjacent bound CRP molecules in the activation of a promoter is an unprecedented observation and might involve molecular mechanisms quite different from those used in the other CRP-controlled promoters.

Mutations that affect transcription and cyclic AMP-CRP regulation of the adenylate cyclase gene (cya) of Salmonella typhimurium.

We studied the expression of the cya promoter(s) in cya-lac fusion strains of Salmonella typhimurium and demonstrated cAMP receptor protein (CRP)-dependent repression by cAMP. Expression of cya was reduced about fourfold in cultures grown in acetate minimal medium as compared to cultures grown in glucose-6-phosphate minimal medium. Expression of cya was also reduced about fourfold by addition of 5 mM cAMP to cultures grown in glucose minimal medium. We constructed in vitro deletion and insertion mutations altering a major cya promoter (P2) and a putative CRP binding site overlapping P2. These mutations were recombined into the chromosome by allele replacement with M13mp::cya recombinant phages and the regulation of the mutant promoters was analyzed. A 4-bp deletion of the CRP binding site and a 4-bp insertion in this site nearly eliminated repression by cAMP. A mutant with the P2 promoter and the CRP binding site both deleted exhibited an 80% reduction in cya expression; the 20% residual expression was insensitive to cAMP repression. This mutant retained a Cya+ phenotype. Taken together, the results establish that the cya gene is transcribed from multiple promoters one of which, P2, is negatively regulated by the cAMP-CRP complex. Correction for the contribution to transcription by the cAMP-CRP nonregulated cya promoters indicates that the P2 promoter is repressed at least eightfold by cAMP-CRP.

Similarities between prokaryotic and eukaryotic cyclic AMP-responsive promoter elements

Organisms as diverse as bacteria and man contain genes that show transcriptional induction when the intracellular concentration of cAMP is increased. This regulated transcriptional response is mediated through specific promoter elements located, in general, upstream from the transcription start site. In Escherichia coli the element responsible for cAMP-mediated transcriptional induction is the binding site for the cAMP-receptor protein (CAP). In mammalian cells the cAMP regulatory element is composed of one or more binding sites for various transcription factors. In many instances the cAMP regulatory element contains binding sites for a family of proteins referred to as ATF. Here we provide evidence that some prokaryotic and mammalian cAMP-response elements are functionally related. First, we show that mammalian ATF binds specifically to some E. coli CAP sites, and conversely E. coli CAP binds specifically to some mammalian ATF sites. Second, we demonstrate that an E. coli CAP binding site can confer cAMP-inducibility onto a mammalian gene when assayed in transfected mammalian cells.

Transcriptional and translational signals of the uidA gene in Escherichia coli K12.

The expression of uidA is negatively controlled by the products of the uidR and uxuR genes and is sensitive to catabolite repression. The locations of the transcriptional and translational signals of uidA were determined using lac gene fusions and S1 mapping experiments. The promoter structure of uidA resembles that of a promoter activated by cAMP receptor protein (CRP); putative control regions are located at positions -10 and -35 (relative to the transcription start site), are separated by more than 17 bp and exhibit poor homology with the normally recognized consensus sequences. Moreover, 80 bp separate the promoter from the translational signals. No CRP binding site was detected in the promoter region of uidA. Two operator sites, 01 and 02, were identified: 01 has a greater affinity for the UidR repressor, whereas 02 has a greater affinity for the UxuR repressor, but the two repressor molecules are able to bind at both the 01 and 02 sites. Analysis of two operator constitutive mutations allowed the location of one of the two UidR repressor binding sites; it contains palindromic units spanning the TaqI site of the uidA control region.

Specific binding of the cAMP receptor protein of Escherichia coli to the lactose operon promoter

The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli. The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8. Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C. The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein. All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride. In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site. Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.

Isolation and characterization of lac fusions to two nitrogen-regulated promoters.

Mud1 (Ap, lac, cts)-mediated fusions to argTr and dhuA, two transport operon promoters in Salmonella typhimurium, were isolated and characterized in order to investigate the regulation of these promoters. Using these fusions we showed that these promoters are under nitrogen regulation and that this effect, as well as the response to a promoter-up mutation in dhuA, is at the transcriptional level. We utilized the fusions to determine that the histidine transport operon does not contain any internal promoters. The fusions were also used to screen the promoters for additional modes of regulation: argTr was found to respond to carbon regulation in addition to nitrogen regulation, while dhuA does not. The argTr promoter contains a sequence with good homology to the consensus sequence determined for the cAMP receptor protein binding site. Neither promoter responds to sulfur or phosphate regulation.

Specific DNA binding of the cAMP receptor protein within the lac operon stabilizes double-stranded DNA in the presence of cAMP.

The effects of varying amounts of cAMP receptor protein (CRP) in the presence and absence of cAMP on the melting and differential melting curves of a 301-bp fragment containing the lac control region in 5 mM Na+ have been investigated. The native 301-bp fragment consists of three cooperatively melting thermalites. At 5 mM Na+, thermalite I (155 bp) has a Tm of 66.4 degrees C and the melting transitions of thermalites II (81 bp) and III (65 bp) are superimposed with a Tm of 61.9 degrees C. The specific DNA target site for CRP and the lac promotor are located within thermalite II. CRP alone exerts no specific effects on the melting of the 301-bp fragment, non-specific DNA binding of CRP resulting in a progressive stabilization of the double-stranded DNA by increasing the number of base pairs melting at a higher Tm in a non-cooperative transition. The cAMP-CRP complex, however, exerts a specific effect with a region of approximately 36 bp, comprising the specific CRP binding site and a neighbouring region of DNA, being stabilized. The appearance of this new cooperatively melting region, known as thermalite IV, is associated with a corresponding decrease in the area of thermalites II/III. The Tm of thermalite IV is 64.4 degrees C, 2.5 degrees C higher than that of thermalites II/III. With two or more cAMP-CRP complexes bound per 301-bp fragment, the stabilization also affects the remaining 110 bp now making up thermalites II/III whose Tm is increased by 1 degrees C to 62.9 degrees C. The implications of these findings for various models of the mode of action of the cAMP-CRP complex are discussed.

Interactions of cAMP receptor protein with the ompA gene, a gene for a major outer membrane protein of Escherichia coli

We have determined the entire DNA sequence of the ompA gene, a gene for a major outer membrane protein of Escherichia coli [ 11. The promoter region of this gene was found to be remarkably homologous with the Zac and gal Pz promoters [2]. Particularly from the fact that both the ompA and gal Pz promoters have the same octanucleotide sequence, T-C-A-C-A-C-T-T in their RNA polymerase recognition site (-35 region), it became relevant to find out if the CAMP receptor protein (CRP) binds to this region of the ompA gene to influence the cellfree transcription of the ompA gene [2]. Here we present the effects of CRP and CAMP on the cell-free transcription of the ompA gene and studies aimed at determining the CRP binding site on the ompA promoter. The results obtained indicate that CRP has a negative effect on the transcription of the ompA gene. We have also rechecked the gels of the DNA sequence of the ompA gene at 7 different positions where the DNAsequence we determined [I] differed from that in [3], including the sequence at the putative transcription termination site, where there is an extremely stable stem-and-loop structure in our sequence but not in theirs.