scholarly journals The AraC/XylS Family Activator RhaS Negatively Autoregulates rhaSR Expression by Preventing Cyclic AMP Receptor Protein Activation

2009 ◽  
Vol 192 (1) ◽  
pp. 225-232 ◽  
Author(s):  
Jason R. Wickstrum ◽  
Jeff M. Skredenske ◽  
Vinitha Balasubramaniam ◽  
Kyle Jones ◽  
Susan M. Egan
Keyword(s):  
Cyclic Amp ◽  
Binding Site ◽  
Binding Sites ◽  
Protein Concentration ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein ◽  
Protein Activation ◽  
High Protein Concentration ◽  
Negative Autoregulation ◽  
The Difference

ABSTRACT The Escherichia coli RhaR protein activates expression of the rhaSR operon in the presence of its effector, l-rhamnose. The resulting RhaS protein (plus l-rhamnose) activates expression of the l-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively. Here, we further investigated our previous finding that rhaS deletion resulted in a threefold increase in rhaSR promoter activity, suggesting RhaS negative autoregulation of rhaSR. We found that RhaS autoregulation required the cyclic AMP receptor protein (CRP) binding site at rhaSR and that RhaS was able to bind to the RhaR binding site at rhaSR. In contrast to the expected repression, we found that in the absence of both RhaR and the CRP binding site at the rhaSR promoter, RhaS activated expression to a level comparable with RhaR activation of the same promoter. However, when the promoter included the RhaR and CRP binding sites, the level of activation by RhaS and CRP was much lower than that by RhaR and CRP, suggesting that CRP could not fully coactivate with RhaS. Taken together, our results indicate that RhaS negative autoregulation involves RhaS competition with RhaR for binding to the RhaR binding site at rhaSR. Although RhaS and RhaR activate rhaSR transcription to similar levels, CRP cannot effectively coactivate with RhaS. Therefore, once RhaS reaches a relatively high protein concentration, presumably sufficient to saturate the RhaS-activated promoters, there will be a decrease in rhaSR transcription. We propose a model in which differential DNA bending by RhaS and RhaR may be the basis for the difference in CRP coactivation.

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

1988 ◽  
Vol 253 (3) ◽  
pp. 801-807 ◽  
Author(s):  
A M Gronenborn ◽  
R Sandulache ◽  
S Gärtner ◽  
G M Clore
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Cyclic Nucleotides ◽  
Binding Pocket ◽  
Receptor Protein ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Cyclic Amp Receptor Protein ◽  
Better Than

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.

scholarly journals Alterations in the binding site of the cyclic AMP receptor protein at the Escherichia coli galactose operon regulatory region

1988 ◽  
Vol 253 (3) ◽  
pp. 809-818 ◽  
Author(s):  
K Gaston ◽  
B Chan ◽  
A Kolb ◽  
J Fox ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Binding Assays ◽  
Cyclic Amp Receptor Protein ◽  
Stimulation Of

Gene manipulation techniques have been used to alter the binding site for the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP) at the regulatory region of the Escherichia coli galactose (gal) operon. The effects of these changes on CRP-dependent stimulation of expression from the galP1 promoter in vivo have been measured, and gel binding assays have been used to measure the affinity of cAMP-CRP for the modified sites. Firstly we have deleted progressively longer sequences from upstream of the gal CRP site in order to locate the functional limit of the site. A deletion to -49, removing the first base that corresponds to the consensus sequence for a CRP binding site, is sufficient to reduce CRP binding and block CRP-dependent stimulation of P1. Secondly, we used synthetic oligonucleotides to invert the asymmetric nucleotide sequence at the gal CRP binding site or to make the sequence symmetric. Inversion of the site has little effect on CRP binding, the architecture of open complexes at P1 revealed by DNAase I footprinting, or the stimulation of transcription from P1. Making the site symmetric increases the affinity for CRP by over 50-fold and leads to increased transcription from P1, whilst hardly altering the DNAase I footprint of open complexes. Our results confirm that the strength of binding of CRP depends on the nature of the site and show that it is this that principally accounts for differences in CRP-dependent stimulation of transcription.

scholarly journals A Cyclic AMP Receptor Protein Mutant That Constitutively Activates an Escherichia coli Promoter Disrupted by an IS5 Insertion

1999 ◽  
Vol 181 (24) ◽  
pp. 7457-7463 ◽  
Author(s):  
Vladimir Podolny ◽  
E. C. C. Lin ◽  
Ann Hochschild
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Regulatory Region ◽  
Constitutive Expression ◽  
Single Amino Acid ◽  
Receptor Protein ◽  
Regulatory Sequences ◽  
Cyclic Amp Receptor Protein ◽  
Coli Promoter

ABSTRACT Previously an Escherichia coli mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phenotype is the result of the constitutive expression of the fucO gene (in the fucAOoperon), which encodes one of the enzymes in the fucose metabolic pathway. The mutant was found to bear an IS5 insertion in the intergenic regulatory region between the divergently orientedfucAO and fucPIK operons. Though expression of the fucAO operon was constitutive, the fucPIKoperon became noninducible such that the mutant could no longer grow on fucose. A fucose-positive revertant which was found to contain a suppressor mutation in the crp gene was selected. Here we identify this crp mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP). We show that the mutant CRP constitutively activates transcription from both the IS5-disrupted and the wild-type fucPIKpromoters, and we identify the CRP-binding site that is required for this activity. Our results show that the fucPIK promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase. For the IS5-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences.

scholarly journals Downregulation of the Escherichia coli guaB Promoter by Upstream-Bound Cyclic AMP Receptor Protein

2009 ◽  
Vol 191 (19) ◽  
pp. 6094-6104 ◽  
Author(s):  
Seyyed I. Husnain ◽  
Stephen J. W. Busby ◽  
Mark S. Thomas
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Cyclic Amp ◽  
Binding Sites ◽  
Progressive Increase ◽  
Receptor Protein ◽  
Lower Growth ◽  
Cyclic Amp Receptor Protein ◽  
Rate Dependent ◽  
A Site

ABSTRACT The Escherichia coli guaB promoter (P guaB ) is responsible for directing transcription of the guaB and guaA genes, which specify the biosynthesis of the nucleotide GMP. P guaB is subject to growth rate-dependent control (GRDC) and possesses an UP element that is required for this regulation. In addition, P guaB contains a discriminator, three binding sites for the nucleoid-associated protein FIS, and putative binding sites for the regulatory proteins DnaA, PurR, and cyclic AMP receptor protein (CRP). Here we show that the CRP-cyclic AMP (cAMP) complex binds to a site located over 100 bp upstream of the guaB transcription start site, where it serves to downregulate P guaB . The CRP-mediated repression of P guaB activity increases in media that support lower growth rates. Inactivation of the crp or cyaA gene or ablation/translocation of the CRP site relieves repression by CRP and results in a loss of GRDC of P guaB . Thus, GRDC of P guaB involves a progressive increase in CRP-mediated repression of the promoter as the growth rate decreases. Our results also suggest that the CRP-cAMP complex does not direct GRDC at P guaB and that at least one other regulatory factor is required for conferring GRDC on this promoter. However, PurR and DnaA are not required for this regulatory mechanism.

scholarly journals The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12.

1990 ◽  
Vol 172 (10) ◽  
pp. 5706-5713 ◽  
Author(s):  
L Søgaard-Andersen ◽  
J Martinussen ◽  
N E Møllegaard ◽  
S R Douthwaite ◽  
P Valentin-Hansen
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein ◽  
Protein Activation ◽  
K 12
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