scholarly journals Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein

FEBS Letters ◽  
1987 ◽  
Vol 219 (1) ◽  
pp. 189-196 ◽  
Author(s):  
Sreenivasan Ponnambalam ◽  
Annick Spassky ◽  
Stephen Busby
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Point Mutation ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein

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.

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 Determinants of the C-Terminal Domain of the Escherichia coli RNA Polymerase α Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters

2002 ◽  
Vol 184 (8) ◽  
pp. 2273-2280 ◽  
Author(s):  
Nigel J. Savery ◽  
Georgina S. Lloyd ◽  
Stephen J. W. Busby ◽  
Mark S. Thomas ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class Ii ◽  
Class I ◽  
Receptor Protein ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Terminal Domain

ABSTRACT Alanine scanning of the Escherichia coli RNA polymerase α subunit C-terminal domain (αCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of αCTD affected class I CRP-dependent transcription from the CC(−61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for αCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in αCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.

Interactions between the Escherichia coli cyclic AMP receptor protein and RNA polymerase at Class II promoters

1993 ◽  
Vol 10 (4) ◽  
pp. 789-797 ◽  
Author(s):  
David West ◽  
Roy Williams ◽  
Virgil Rhodius ◽  
Andrew Bell ◽  
Naveen Sharma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class Ii ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein

scholarly journals Interactions between the cyclic AMP receptor protein and the Alpha subunit of RNA polymerase at the Escherichia Coli galactose operon P1 Promoter

1994 ◽  
Vol 22 (21) ◽  
pp. 4375-4380 ◽  
Author(s):  
Adrian Attey ◽  
Tamara Belyaeva ◽  
Nigel savery ◽  
Jim Hoggett ◽  
Nobuyuki Fujita ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Alpha Subunit ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein

scholarly journals Positioning of Region 4 of the Escherichia coli RNA Polymerase ς70 Subunit by a Transcription Activator

2000 ◽  
Vol 182 (10) ◽  
pp. 2982-2984 ◽  
Author(s):  
Jonathan A. Bown ◽  
Annie Kolb ◽  
Claude F. Meares ◽  
Akira Ishihama ◽  
Stephen D. Minchin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Dna Cleavage ◽  
Transcription Activation ◽  
Receptor Protein ◽  
Transcription Activator ◽  
Cyclic Amp Receptor Protein ◽  
Conformation Changes ◽  
Major Conformation

ABSTRACT A DNA cleavage reagent, specifically tethered to residue 581 of theEscherichia coli RNA polymerase ς70 subunit, has been used to investigate the location of ς70 region 4 in different complexes at the galp 1 promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at thegalp 1 promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of ς70 region 4.

Sign in / Sign up

Export Citation Format

Share Document