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  • 1
    Electronic Resource
    Electronic Resource
    Dissection of a surface-exposed portion of the cAMP–CRP complex that mediates transcription activation and repression (1999)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 32 (1999), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Escherichia coli cAMP receptor protein (CRP) is essential for the activation and repression of transcription initiation at promoters in the CytR regulon. CRP performs these activities by making direct protein–protein interactions to the α-subunits of RNA polymerase and to the CytR regulator. Strikingly, it has been shown that amino acids of CRP that are critical for communication with the two partner proteins are located in close proximity on the surface of CRP. Here, we have dissected this surface in order to pinpoint the ‘repression region’ of CRP and to assess whether it overlaps with the characterized ‘activating region’. Our results established that residues 12, 13, 17, 105, 108 and 110 are essential for the interaction with CytR and confirmed that ‘activating region’ 2 of CRP is made up of residues 19, 21 and 101. In the crystallographic structure of the CRP–DNA complex, the two sets of determinants are located immediately adjacent to each other forming a consecutive surface-exposed patch. The ‘repression region’ is chemically complementary to the characterized region on CytR that is essential for protein–protein communication to CRP. Moreover, the results provide insight into the mechanism by which CytR might prevent CRP-mediated transcription.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01362.x
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  • 2
    Electronic Resource
    Electronic Resource
    DNA-binding characteristics of the Escherichia coli CytR regulator: a relaxed spacing requirement between operator half-sites is provided by a flexible, unstructured interdomain linker (1998)
    Oxford BSL : Blackwell Publishing Ltd
    Molecular microbiology 27 (1998), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Escherichia coli CytR regulator belongs to the LacI family of sequence-specific DNA-binding proteins and prevents CRP-mediated transcription in the CytR regulon. Unlike the other members of this protein family, CytR binds with only modest affinity to its operators and transcription repression thus relies on the formation of nucleoprotein complexes with the cAMP–CRP complex. Moreover, CytR exhibits a rotational and translational flexibility in operator binding that is unprecedented in the LacI family. In this report we examined the effect of changing the spacing between CytR half-operators on CytR regulation in vivo and on CytR binding in vitro. Maximum repression was seen with the short spacing variants: repression peaks when the half-operators lie on the same face of the DNA helix. Repression was retained for most spacing variants with centre separations of half-operators ≤ 3 helical turns. Our data confirm and extend the view that CytR is a highly flexible DNA binder that can adapt many different conformations for co-operative binding with CRP. Furthermore, limited proteolysis of radiolabelled CytR protein showed that the interdomain linker connecting the DNA binding domains and the core part of CytR does not become structured upon DNA binding. We conclude that CytR does not use hinge α-helices for minor groove recognition. Rather, CytR possesses a highly flexible interdomain linker that allows it to form complexes with CRP at promoters with quite different architecture.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00655.x
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  • 3
    Electronic Resource
    Electronic Resource
    Tandem DNA-bound cAMP-CRP complexes are required for transcriptional repression of the deoP2 promoter by the CytR repressor in Escherichia coli (1990)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 4 (1990), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have studied the deoP2 promoter in Escherichia coli to define features important for its interaction with the CytR repressor. As is characteristic for CytR-regulated promoters, deoP2 encodes tandem binding sites for the activating complex cAMP-CRP. One of these sites, CRP-1, overlaps the - 35 region, and is sufficient for activation; the second site, CRP-2, centred around-93, is indispensable for repression. Here we demonstrate, by means of in vivo titration, that CytR interaction with deoP2 depends not only on CRP-2, but also on CRP-1 and the length and possibly the sequence separating these two sites. Also, point mutations in either CRP site reduce or abolish CytR titration; however, no co-operativity is observed in the interaction of CytR with the two CRP binding sites. Furthermore, the reduction in CytR titration parallels the reduction in binding of cAMP-CRP to the mutated CRP sites in vitro. These observations are not easily explained by current models for the action of prokaryotic repressors; instead we favour a model in which the interaction of CytR with deoP2 depends on the presence of tandem DNA-bound cAMP–CRP complexes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1990.tb02071.x
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  • 4
    Electronic Resource
    Electronic Resource
    Transcriptional regulation of the cytR repressor gene of Escherichia coli: autoregulation and positive control by the cAMP/CAP complex (1990)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 4 (1990), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Escherichia coli cytR-encoded repressor protein (CytR) controls the expression of several genes involved in nucleoside and deoxynucleoside uptake and metabolism. The cytR promoter was identified by determining the transcriptional initiation site of the cytR gene. A chromosomal cytR-lacZ+ operon fusion was isolated and used to study the regulation of cytR. We show that cytR expression is negatively controlled by the CytR protein and positively affected by the cAMP/CAP complex. Foot printing studies with purified CAP protein revealed two CAP binding sites upstream of the cytR promoter. A previousty described mutation (cytR*) in the cloned cytR gene, which results in the phenotypic suppression of a CytR operator mutation in the tsx P2 promoter, was analysed. DN A sequence analysis of the cytR* mutation revealed a G-C to an A-T base pair transition at position -34 bp relative to the translational initiation site of cytR. This point mutation activates a cryptic promoter that is stronger than the wild-type cytR promoter and leads to overproduction of the CytR repressor.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1990.tb00614.x
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  • 5
    Electronic Resource
    Electronic Resource
    CRP/cAMP- and CytR-regulated promoters in Escherichia coli K12: the cdd promoter (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Transcriptional regulation of the deoP2 promoter by the cyclic AMP/cyclic AMP receptor protein complex (cAMP/CRP) and the CytR repressor requires two high-affinity CRP targets located around -41 and -93 bp preceding the start site for transcription. Here we report the structure of cddP, another CRP/CytR-regulated promoter. In common with what was found in deo, the cdd promoter also contains multiple CRP targets. Thus, using the DNasel footprinting procedure, tandem CRP binding sites were identified around -41 and -93. These findings support a general model for CytR binding and CytR regulation, in which (i) CytR and the CRP/cAMP complex bind to similar or Identical targets, (ii) two or more targets are necessary for proper binding of CytR to a promoter region, and (iii) CytR represses transcription by antagonizing cAMP/CRP activation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1989.tb00120.x
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  • 6
    Electronic Resource
    Electronic Resource
    Design of camp–CRP-activated promoters in Escherichia coli (1991)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 5 (1991), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have studied the doeP2 promoter of Escherichia coli to define features that are required for optimal activation by the complex of adenosine 3′, 5′ monophosphate (cAMP) and the cAMP receptor protein (CRP). Systematic mutagenesis of deoP2 shows that the distance between the CRP site and the -10 hexamer is the crucial factor in determining whether the promoter is activated by camp–CRP. Based on these observations, we propose that camp–CRP-activated promoters can be created by correctly aligning a CRP target and a - 10 hexamer. This idea has been successfully tested by converting both a CRP-in-dependent promoter and a sequence resembling the consensus -10 hexamer to strongly camp–CRP-activated promoters.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1991.tb02126.x
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  • 7
    Electronic Resource
    Electronic Resource
    A novel function of the cAMP-CRP complex in Escherichia coli: cAMP-CRP functions as an adaptor for the CytR repressor in the deo operon (1991)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 5 (1991), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Unlike classical 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1991.tb00772.x
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  • 8
    Electronic Resource
    Electronic Resource
    Analysis of the tsx gene, which encodes a nucleoside-specific channel-forming protein (Tsx) in the outer membrane of Escherichia coli
    Amsterdam : Elsevier
    Gene 96 (1990), S. 59-65 
    Details
    ISSN: 0378-1119
    Keywords: Nucleoside uptake ; bacteriophage receptor ; colicin K receptor ; gene regulation ; promoters ; recombinant DNA
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
    URL: http://linkinghub.elsevier.com/retrieve/pii/0378-1119(90)90341-N
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  • 9
    Electronic Resource
    Electronic Resource
    A second purine nucleoside phosphorylase in Escherichia coli K-12 (1980)
    Springer
    Molecular genetics and genomics 179 (1980), S. 331-340 
    Details
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Enzyme purification studies have indicated the existence in E. coli of only a single species of purine nucleoside phosphorylase. Strains mutated in the gene (deoD) specifying this enzyme cannot grow on purine nucleosides such as inosine, adenosine and guanosine as carbon source. We have selected secondary-site revertants of a deoD strain by plating on adenosine or inosine as carbon source and we have shown that the site of the mutation enabling growth on adenosine or inosine in these revertants, termed xapR, lies between nupC and ptsI at 51 min, almost exactly 180° from the deoD gene on the E. coli chromosome. In some xapR mutants there is a constitutive synthesis of a second purine nucleoside phosphorylase; in other xapR mutants, this enzyme is induced by inosine. The properties of this enzyme in the xapR mutants are very similar to that of xanthosine phosphorylase found in wild-type cells induced with xanthosine, and we thus consider that xapR mutants are altered in the regulation of xanthosine phosphorylase. From an xapR strain mutants were isolated which lacked this second purine nucleoside phosphorylase. The site of this mutation, xap, was 90% co-transducible with xapR. Such strains could not grow on xanthosine as sole carbon source. The rate of mutation to xapR − was very low (3x10-8). Also studies with an F-prime covering the xapR + gene revealed that xapR2 was partially dominant to xapR +. We therefore suggest that the xapR + gene product is an inducer protein for the gene specifying xanthosine phosphorylase, which is inactive until converted by the inducer into a form able to switch on the operon, i.e. there is positive control of the xap gene.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00425461
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