CRP Interacts Specifically With Sxy to Activate Transcription in Escherichia coli (original) (raw)
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The Sxy (TfoX) protein is required for expression of a distinct subset of the genes regulated by the cAMP receptor protein (CRP) in the model organisms Escherichia coli, Haemophilus influenzae, and Vibrio cholerae. Genetic studies have established that CRP and Sxy co-activate transcription at gene promoters containing DNA binding sites called CRP-S sites. In contrast, CRP acts without Sxy at gene promoters containing canonical CRP-N sites, suggesting that Sxy makes physical contacts with CRP and/or DNA to assist in transcriptional activation at CRP-S promoters. Despite growing interest in Sxy’s activity as a transcription factor, Sxy remains poorly characterized due to a lack of reliable phenotypes in E. coli. Experiments are further hampered by growth inhibition and formation of inclusion bodies when Sxy is overexpressed. In this study we applied diverse phenotypic and molecular assays to test for postulated Sxy functions and interactions. Mutations in conserved regions of Sxy and ...
The Camp-CRP Cytr Nucleoprotein Complex in Escherichia-Coli - is Cytr More Than a Local Regulator
Journal of Cellular Biochemistry, 1995
Transcription initiation at CytR regulated promoters in Escherichia coli is controlled by a combinatorial regulatory system in which the cAMP receptor protein (CRP) functions as both an activator and a co-repressor. By combining genetic studies and footprinting analyses, we demonstrate that regulated expression of the CytR controlled cdd promoter requires three CRP-binding sites: a high affinity site (CRP-1) and two overlapping low affinity sites (CRP-2 and CRP-3) centred at positions-41,-91 and-93, respectively. In the absence of CytR, cAMP-CRP interacts at one set of sites (CRP-1 and CRP-2) and both of these binding sites are required for full promoter activation. In the presence of CytR, however, the two regulators bind cooperatively to cddP forming a nucleoprotein complex in which cAMP-CRP binds to CRP-1 and CRP-3 and CytR occupies the sequence between these sites. Thus, association of the two regulators involves a repositioning of the cAMP-CRP complex. Moreover, mutant cdd promoters in which CRP-2 and CRP-3 have been deleted are partially regulated by CytR, and cAMP-CRP and CytR still bind cooperatively to these promoters. These findings provide clues to an understanding of how cAMP-CRP and CytR interact at a structurally diverse set of promoters.
Molecular Microbiology, 1991
Unlike cfassicat bacterial repressors, the CytR repressor of Escherichia coli cannot independently regulate gene expression. Here we show that CytR binding to the deoP2 promoter relies on interaction with the master gene regulatory protein, CRP, and, furthermore, that cAMP-CRP and CytR bind co-operatively to deoP2. Using mutant promoters we show that tandem, properly spaced DNA-bound cAMP-CRP complexes are required for this co-operative binding. These data suggest that CytR forms a bridge between tandem cAMP-CRP complexes, and that cAMP-CRP ftjnctions as an adaptor for CytR. The implications of this new version of negative control in E. coli on bacterial gene expression and on combinatorial gene regulation in higher organisms are discussed.
The EMBO journal, 1992
Transcription initiation at CytR regulated promoters in Escherichia coli is controlled by a combinatorial regulatory system in which the cAMP receptor protein (CRP) functions as both an activator and a co-repressor. By combining genetic studies and footprinting analyses, we demonstrate that regulated expression of the CytR controlled cdd promoter requires three CRP-binding sites: a high affinity site (CRP-1) and two overlapping low affinity sites (CRP-2 and CRP-3) centred at positions -41, -91 and -93, respectively. In the absence of CytR, cAMP-CRP interacts at one set of sites (CRP-1 and CRP-2) and both of these binding sites are required for full promoter activation. In the presence of CytR, however, the two regulators bind cooperatively to cddP forming a nucleoprotein complex in which cAMP-CRP binds to CRP-1 and CRP-3 and CytR occupies the sequence between these sites. Thus, association of the two regulators involves a repositioning of the cAMP-CRP complex. Moreover, mutant cdd p...
J Mol Biol, 1998
The general stress-induced sigma subunit s S of Escherichia coli RNA polymerase is closely related to the vegetative sigma factor s 70. In view of their very similar promoter speci®city in vitro, it is unclear how sigma factor selectivity in the expression of s S-dependent genes is generated in vivo. The csiD gene is such a strongly s S-dependent gene. In contrast to s S , which is induced in response to many different stresses, csiD, whose expression is driven from a single promoter, is induced by carbon starvation only. To our knowledge, the csiD promoter is the ®rst characterized promoter which is not only exclusively dependent on s S-containing RNA polymerase (Es S), but also requires an activator, cAMP-CRP. In addition, leucine-responsive regulatory protein (Lrp) acts as a positive modulator of csiD expression. Also in vitro, Es S is more ef®cient than Es 70 in csiD promoter binding, open complex formation and runoff transcription, which might be due to the poor match of the csiD À35 region to the s 70 consensus and to transcription by Es S being less dependent on contacts in this region. By DNase I protection experiments, a cAMP-CRP binding site centered at À68.5 nucleotides upstream of the csiD transcriptional start site was identi®ed. While cAMP-CRP stimulates Es 70 binding, it does not promote open complex formation by Es 70 , but does so in conjunction with Es S. With linear templates, cAMP-CRP signi®cantly stimulates Es S-mediated in vitro transcription, whereas transcription by Es 70 is negligible and hardly stimulated by cAMP-CRP. These ®ndings may re¯ect different or less stringent positional requirements for an activator site for Es S than for Es 70 , and indicate that cAMP-CRP contributes to sigma factor selectivity at the csiD promoter. In vitro transcription experiments with supercoiled templates, however, revealed signi®cant cAMP-CRP-stimulated transcription also by Es 70. Yet, under these conditions, H-NS was found to restore Es S speci®city by strongly interfering with cAMP-CRP/Es 70dependent transcription. Lrp strongly and cooperatively binds to multiple sites located between positions À14 and À102 (in a way that suggests DNA wrapping around multiple Lrp molecules) and moderately stimulates in vitro transcription, especially with Es S. In summary, we conclude that the csiD promoter has an intrinsic preference for Es S , but that also protein factors such as cAMP-CRP, Lrp and probably H-NS as well as DNA conformation contribute to its strong Es S selectivity. Furthermore, this strong Es S preference in combination with a requirement for high concentrations of the essential activator cAMP-CRP
Journal of Molecular Biology, 1997
In CytR regulated promoters in Escherichia coli, the cAMP-CRP complex acts as a transcriptional activator as well as a co-repressor for the CytR protein. Repression by CytR depends on the formation of nucleoprotein complexes in which CytR binds cooperatively to the DNA with one or two cAMP-CRP complexes. Here, we demonstrate that in order to establish CytR regulation in a cAMP-CRP dependent class II promoter with a single CRP site (CRP site centred around position À40.5) in which the CytR operator is located upstream of the CRP site, high af®nity binding sites for both regulators are required. The ef®ciency of CytR regulation was observed to be modulated by RNA polymerase (RNAP)-promoter interactions. Speci®cally, in class II promoters with a single CRP site, the ef®ciency of CytR regulation was found to correlate inversely with cAMP-CRP independent promoter activity. These observations can be reconciled in a competition model for CytR regulation in which CytR and RNAP compete for cooperative binding with cAMP-CRP to the promoters in vivo. In this model, two mutually exclusive ternary complexes can be formed: a CytR/cAMP-CRP/promoter repression complex and an RNAP/cAMP-CRP/promoter activation complex. Thus, CytR regulation critically depends on formation of a repression complex that binds the promoter with suf®ciently high af®nity to exclude formation of the competing activation complex. We suggest that the transition from repression to activation involves a switch in the protein-protein interactions made by cAMP-CRP from CytR to RNAP. On the basis of the regulatory features of the promoters analysed here, we speculate about the advantages offered by the structural complexity of natural CytR/cAMP-CRP regulated promoters.
Cell Biochemistry and Function, 2008
The cyclic AMP receptor protein (CRP) of Escherichia coli regulates the activity of more than 150 genes. Allosteric changes in CRP structure accompanied by cAMP binding, initiate transcription through protein binding to specific DNA sequences. Initially, researchers proposed a two-site cAMP-binding model for CRP-dependent transcription activation since biophysical methods showed two transitions during titration experiments. Three conformational states were considered; apo-CRP, CRP:(cAMP) 1 and CRP:(cAMP) 2 , and CRP:(cAMP) 1 was proposed as the active form in this initial model. X-ray data indicated an anti conformation and in contrast NMR experiments suggested a syn conformation for bound cAMPs. For years this paradigm about ligand conformation has been ambiguous. When CRP was crystallized with four bound cAMP in the last decade, two cAMPs were assigned to syn and the other two to anti conformations. Again three conformational states were suggested; apo-CRP, CRP:(cAMP) 2 , and CRP:(cAMP) 4 . This new structure changed the view of CRP allosteric activation from a two-site model to a four-site model in the literature and the new model has been supported by biochemical and genetic data so far. According to the accepted model, binding of the first two cAMP molecules displays positive cooperativity, however, binding of the last two cAMP molecules shows negative cooperativity. This resolved the conflict between dynamic and static experimental observations. However, this new model cannot explain the initiation mechanism as previously proposed because functionally active CRP has only one cAMP equivalent. Gene regulation and transcription factors are involved in regulating both prokaryotic and eukaryotic metabolism. Although gene regulation and expression are much more complex in eukaryotes, CRP-mediated transcription initiation is a model of general interest to life sciences and medicine. Therefore, the aim of this review is to summarize recent works and developments on the cAMP-dependent CRP activation mechanism in E. coli.
The Journal of biological chemistry, 1991
Plasmids were constructed that contain deletions in the stem region of the presumed rho-independent terminator of the cloned crp gene of Escherichia coli. The level of cyclic AMP binding activity and the amount of CRP in cells harboring the deletion plasmids were found to be significantly lower than those in cells harboring the wild-type crp plasmid. Quantitative S1 assays indicated that the steady-state levels of crp mRNA were markedly reduced in cells harboring the deletion plasmids. Evidence was also presented to show that the crp mRNAs produced from deletion plasmids are less stable than that from the intact crp gene. In vitro transcription assays revealed that the putative crp terminator is indeed a rho-independent terminator. Using the galK expression system and Northern blot analysis we showed that the crp terminator is functional in vivo. Moreover it was shown that the deletion mutations in the stem region of the crp terminator cause a significant readthrough. We conclude th...
Biochemistry, 1996
The interactions between the cAMP receptor protein (CRP) and RNA polymerase during transcriptional activation at the Escherichia coli malT promoter have been analyzed using a combination of footprinting methods. We show that a closed complex is formed at this promoter in the absence of activator and that CRP merely stabilizes the open complex. The R-subunits of the RNA polymerase are involved in this effect as shown by KMnO 4 footprinting. The open complex formed in the presence of CRP is structurally identical to the one found at a CRP-independent promoter up-mutant. UV-laser footprinting yields distinct signals for the different protein-DNA interactions within the complex and for interactions between CRP and RNA polymerase. We monitor these signals in promoter variants that place the CRP binding site at different distances upstream of the start site of transcription. Signals within the core promoter region, as well as those located just upstream of the-35 hexamer, are unaffected by the position of the CRP binding site. Contacts of RNA polymerase with the upstream promoter region change in a mutant RNA polymerase containing a truncated R-subunit. We conclude that at least one of the R-subunits of RNA polymerase binds to DNA upstream of the-35 hexamer and that this interaction is unaffected by the position of the CRP binding site. We discuss models that account for the different activities of CRP in transcriptional activation as a function of promoter geometry.
Molecular Microbiology, 1991
The environmentally activated algD promoter of Pseudomonas aeruginosa has been shown to be influenced by DNA supercotling. It is believed that protein-induced bending or looping is required for this activation. We studied the role of Escherichia coli cAMP-CRP on a/^D promoter activation in E. coli and show that a functional CRP is required for this activation. We also demonstrate that the algD promoter is sensitive to glucose repression both in E. coli and P. aeruginosa. Deletion of a putative consensus CRP binding sequence upstream of the algD promoter renders the promoter non-responsive to glucose repression. The involvement of cAMP-CRP complex in the activation of the afgD promoter in E. coU has been demonstrated directfy through binding of a 255 base pair DNA fragment containing the putative consensus CRP binding sequence. Other fragments, upstream or downstream but without any consensus CRP binding sequence, did not show any binding with CRP. A CRP-like analogue, similar to that in Xanthomonas campestris, but capable of activating genes without forming a complex with cAMP, is believed to allow glucose repression in P. aeruginosa. . 'For correspondence. Tel. WJ2J 996 4586, Fax (312) 996 6415.