Mutations in the Escherichia coli operon that define two promoters and the binding site of the cyclic AMP receptor protein

1982 ◽  
Vol 154 (2) ◽  
pp. 211-227 ◽  
Author(s):  
Stephen Busby ◽  
Hiroji Aiba ◽  
Benoît De Crombrugghe
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein

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.

Mannose utilization in Escherichia coli requires cyclic AMP but not an exogenous inducer

1980 ◽  
Vol 26 (12) ◽  
pp. 1508-1511 ◽  
Author(s):  
Ann D. E. Fraser ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Sole Carbon Source ◽  
Receptor Protein ◽  
Deficient Mutant ◽  
Phosphotransferase System ◽  
Cyclic Amp Receptor Protein ◽  
Cyclic Monophosphate

It has not been clarified whether the utilization of mannose by Escherichia coli requires adenosine 3′,5′-cyclic monophosphate (cyclic AMP). Using an adenylyl cyclase deficient mutant (CA8306B) and a cyclic AMP receptor protein (CRP) deficient mutant (5333B) we have shown that the utilization of mannose is dependent on the cyclic AMP–CRP complex. 2-Deoxyglucose (DG) is a nonmetabolizable glucose analog specific for the phosphotransferase system (PTS) which transports mannose (termed here PTSM). Growth of CA8306B on glycerol is unaffected by addition of the analog, whereas growth of the strain on glycerol plus cyclic AMP ceases im mediately upon addition of DG. These results suggest that the formation of PTSM is dependent on cyclic AMP. In addition, CA8306B grown on glycerol plus cyclic AMP can immediately utilize mannose when transferred to a medium containing mannose as a sole carbon source, whereas the same strain grown on glycerol without cyclic AMP cannot utilize mannose when so transferred. These results suggest that the formation of PTSM does not require an exogenous inducer.

scholarly journals Cyclic AMP Receptor Protein-Dependent Activation of the Escherichia coli acsP2 Promoter by a Synergistic Class III Mechanism

2003 ◽  
Vol 185 (17) ◽  
pp. 5148-5157 ◽  
Author(s):  
Christine M. Beatty ◽  
Douglas F. Browning ◽  
Stephen J. W. Busby ◽  
Alan J. Wolfe
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class I ◽  
Receptor Protein ◽  
Class Iii ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Rna Polymerase Holoenzyme

ABSTRACT The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by “focusing” RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions −69.5 and −122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the α subunit of RNA polymerase holoenzyme (α-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the α-CTD. Other surface-exposed residues in the α-CTD contributed to acs transcription, suggesting that the α-CTD may interact with at least one protein other than CRP.

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